Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins.

The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and beta-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins.

Ago-TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps.

MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.

Molecular analysis of the human $\gamma$-chain T cell receptor genes

In our studies we have focused on the issue of variability and diversity of the $\gamma$ (or $\delta)$ chain T cell receptor (TCR) genes by studying cDNA transcripts in peripheral blood mononuclear cells or $\gamma\delta$ TCR+ T cell clones. The significance of these studies lies in the better understanding of the molecular biology of the $\gamma\delta$ T cell receptor as well as in answering the question whether certain molecular forms predominate in $\gamma\delta$ T cells exhibiting specific immunologic functions. We establish that certain $\gamma$-chain TCR genes exhibit particular patterns of rearrangements in cDNA transcripts in normal individuals. V$\gamma$I subgroup were shown to preferentially rearrange to J$\gamma$2C$\gamma$2 gene segments. These preferential VJC rearrangements, may have implications regarding the potential for diversity and polymorphism of the $\gamma$-chain TCR gene. In addition, the preferential association of V$\gamma$I genes with J$\gamma$2C$\gamma$2, which encode a non-disulfide-linked $\gamma\delta$ TCR, suggests that $\gamma$ chains utilizing V$\gamma$I are predominantly expressed as non-disulfide-linked $\gamma\delta$ TCR heterodimers. The implications of this type of expression remain to be determined. We identified two alternative splicing events of the $\gamma$-chain TCR genes occurring in high frequency in all the normal individuals examined. These events may suggest additional mechanisms of regulation and control as well as diversification of $\gamma\delta$ TCR gene expression. The question whether particular forms of $\gamma$ or $\delta$-chain TCR genes are involved in HLA Class I recognition by specific $\gamma\delta$ cytotoxic T cell clones was addressed. Our results indicated that the T cell clones expressed identical $\gamma$ but distinct $\delta$-chains suggesting that the specificity for recognition of HLA-A2 or HLA-A3 may be conferred by the $\delta$-chain TCR. The issue of the degree of diversity and polymorphism of the $\delta$-chain TCR genes in a patient with a primary immunodeficiency (Omenn's syndrome) was addressed. A limited pattern of rearrangements in peripheral blood transcripts was found, suggesting that a limited $\gamma\delta$ TCR repertoire may be expressed in this particular primary immunodeficiency syndrome. Overall, our findings suggest that $\delta$-chain TCR genes exhibit the potential for significant diversity and that there are certain preferential patterns of expression that may be associated with particular immunologic functions. ^

The cellular and molecular involvement of glutathione in cisplatin-induced apoptosis

The goal of this study was to investigate the cellular and molecular mechanisms by which glutathione (GSH) is involved in the process of apoptosis induced by cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] in the HL60 human promyelocytic leukemia cell line. The data show that during the onset or induction of apoptosis, GSH levels in cisplatin-treated cells increased 50% compared to control cells. The increase in intracellular GSH was associated with enhanced expression of γ-glutamylcysteine synthetase (γ-GCS), the enzyme that catalyzes the rate- limiting step in the biosynthesis of glutathione. After depletion of intracellular GSH with D,L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of γ-GCS, biochemical and morphological analysis revealed that the mechanism of cell death had switched from apoptosis to necrosis. In contrast, when intracellular GSH was elevated by exposure of cells to a GSH-ethyl-ester and then treatment with cisplatin, no change in the induction and kinetics of apoptosis were observed. However, when cells were exposed to cisplatin before intracellular GSH levels were increased, apoptosis was observed to occur 6 hours earlier compared to cells without GSH elevation. To further examine the molecular aspects of these effects of GSH on the apoptotic process, changes in the expression of bcl-2 and bax, were investigated in cells with depleted and elevated GSH. Using reverse transcription polymerase chain reaction, no significant change in the expression of bcl-2 gene transcripts was observed in cells in either the GSH depleted or elevated state; however, a 75% reduction in GSH resulted in a 40% decrease in the expression of bax gene transcripts. In contrast, a 6-fold increase in GSH increased the expression of bax by 3-fold relative to controls. Similar results were obtained for bax gene expression and protein synthesis by northern analysis and immunoprecipitation, respectively. These results suggest that GSH serves a dual role in the apoptotic process. The first role which is indirect, involves the protection of the cell from extensive damage following exposure to a specific toxicant so as to prevent death by necrosis, possibly by interacting with the DNA damaging agent and/or its active metabolites. The second role involves a direct involvement of GSH in the apoptotic process that includes upregulation of bax expression. ^

Extended duration of torsional loading reduced the survival of the NP cells of the intervertebral disc

Introduction Previous studies on the influence of torsion and combined torsion-compression loading revealed a positive effect on the cell viability when a repetitive short-term torsion was applied at a physiological magnitude to intervertebral disc organ culture.1 However, after an extended period (8 hours) of combined torsion-compression loading, substantial cell death was detected in the nucleus pulposus (NP).2 In this follow-up study, we aimed to investigate the relationship, if any, between the duration of torsion applied to the intervertebral disc (IVD) and the level of NP cell viability. Materials and Methods Bovine caudal discs were harvested and cultured in a custom-built multiaxis dynamic loading bioreactor.2 Torsion (± 2 degrees) was applied to the samples at a frequency of 0.2 Hz. Torsion was applied for durations of 0, 1, 4, and 8 h/d, repeated over 7 days. After the last day of loading, disc tissue was dissected for analysis of cell viability and gene expression. Results Disc NP cell viability remained above 85% after torsional loading for 0, 1, or 4 h/d. Viability was statistical significantly reduced to below 70% when torsion was applied for 8 h/d (p = 0.03) (Table 1). The daily duration of torsional loading did not affect the AF cell viability (> 80% for all loading durations). The trend of collagen 2 gene upregulation and matrix metalloproteases 13 downregulation with an increasing duration of torsion was observed in both NP and AF (Fig. 1).Conclusion We have demonstrated that an extended duration of torsion could inhibit the survival of NP cells within the IVD in organ culture. Acknowledgments Funds from the Orthopedic Department of the Insel University Hospital of Bern and a private donation from Prof. Dr. Paul Heini, Spine Surgeon, Sonnenhof Clinic Bern were received to support this work. Disclosure of Interest None declared References References 1 Chan SC, Ferguson SJ, Wuertz K, Gantenbein-Ritter B. Biological response of the intervertebral disc to repetitive short-term cyclic torsion. Spine 2011;36(24):2021–2030 2 Chan SC, Walser J, Käppeli P, Shamsollahi MJ, Ferguson SJ, Gantenbein-Ritter B. Region specific response of intervertebral disc cells to complex dynamic loading: an organ culture study using a dynamic torsion-compression bioreactor. PLoS ONE 2013;8(8):e72489

A heteromeric potassium channel involved in the modulation of the plasma membrane potential is essential for the survival of African trypanosomes.

Discovery of novel drug targets may lead to improved treatment of trypanosomiasis. We characterize here 2 gene products of Trypanosoma brucei that are essential for the growth of bloodstream form (BSF) parasites, as shown by RNA interference (RNAi)-mediated down-regulation of the individual mRNAs. The primary sequences of the 2 proteins--protein encoded by gene Tb927.1.4450 (TbK1) and protein encoded by gene Tb927.9.4820 (TbK2)--indicate that both belong to the family of putative, Ca(2+)-activated potassium channels. The proteins were expressed in Xenopus laevis oocytes and their functions investigated by use of electrophysiological techniques. Only combined expression of TbK1 and TbK2 results in the formation of sizeable currents, indicating that these proteins probably assemble into a heteromeric ion channel. The current mediated by this channel shows little time and voltage dependence and displays a permeability ratio of K(+)/Na(+) of >20. The known potassium channel blocker barium inhibits this channel with a half-maximal inhibitory concentration (IC50) of 98 ± 15 μM. The membrane potential of trypanosomes was measured with a fluorescent dye. Individual RNAi-mediated down-regulation of TbK1 or TbK2 eliminates a potassium conductance in the plasma membrane of BSF. Thus, this heteromeric potassium channel is involved in the modulation of the plasma membrane potential and represents a novel drug target in T. brucei.

Role of the transcription factor activator protein-2alpha in prostate carcinogenesis

Activator protein 2α (AP-2) is a transcription factor known to play a crucial role in the progression of malignant melanoma, colorectal carcinoma, and breast cancer. Several AP-2 target genes are known to be deregulated in prostate cancer, therefore, we hypothesize that loss AP-2 expression plays a causal role in prostate carcinogenesis. Immunofluorescent staining for AP-2 of 30 radical prostatectomy specimens demonstrated that while AP-2 was highly expressed in normal prostate epithelium, its expression was lost in most cases of high grade prostatic intraepithelial neoplasia (PIN), and all cases of prostate cancer studied. Additional analyses demonstrated that AP-2 was associated with normal luminal differentiation and it was not expressed in the basal cell layer. In cell lines, AP-2 was strongly expressed in immortalized normal prostate epithelial cells, whereas low expression was observed in the LNCaP, LNCaP-LN3, and PC3M-LN4 prostate cancer cell lines. Transfection of the highly tumorigenic and metastatic cell line PC3M-LN4 with the AP-2 gene significantly decreased tumor growth in the prostate of nude mice (p = 0.032) and inhibited metastases to the lymph nodes. Moreover, transfection of the low tumorigenic, low metastatic cell line LNCaP-LN3 with full length AP-2; resulted in complete inhibition of tumor incidence in the AP-2 transfectants (0/19) vs. neo control (10/16). A potential mechanism for this loss of tumorigenicity was the modulation of gene expression in prostate cancer cells that mimicked the normal phenotype. Analysis of differential expression between neo control- and AP-2-transfected cells in vitro and in tumors demonstrated low VEGF expression in AP-2 transfectants. We further demonstrated that AP-2 acted as a transcriptional repressor of the VEGF promoter by binding to a GC-rich region located between −88 and −66. This region contains an AP-2 consensus element overlapping two Sp1 consensus elements. We found that Sp3 and AP-2 bound to this region in a mutually exclusive manner to promote activation or repression. Increased VEGF expression has been observed in high grade PIN and in prostate cancer. Here we provide evidence that this early molecular change could be a result of loss of AP-2 expression in the prostatic epithelium. ^

Convergent etiology of Eker rat and human uterine leiomyoma

The Eker rat model has allowed researchers the unique opportunity to study the tumorigenesis of spontaneously occurring uterine leiomyoma. Animals in this line harbor a germline mutation in the tuberous sclerosis complex-2 (Tsc-2) tumor suppressor gene and develop uterine leiomyomas at a rate of ∼65%. Primary leiomyomas obtained from humans and Eker rats along with Eker-derived leiomyoma cell lines were used in studies described herein to determine the effect of PPARγ ligand treatment on the proliferation of this cell type and to determine the role of tuberin and p27Kip1 in the etiology of this tumor type. Treatment of leiomyoma cells of human and rat origin with PPARγ-activating compounds resulted in decreased proliferation. Additionally, PPARγ ligands inhibited estrogen-dependent gene transactivation in Eker-derived leiomyoma cells suggesting that nuclear receptor cross-talk may exist between PPAR and the ER and may be responsible for the inhibition of proliferation in this cell type. Loss of tuberin, the product of the TSC-2 gene, is associated with Eker rat leiomyoma development while the role of this tumor suppressor in human leiomyoma development is unknown. Data herein show that tuberin expression is diminished in 25% of human leiomyomas tested. Additionally, we observed diminished p27 Kip1 expression in 80% of human uterine leiomyomas compared to normal myometrium. Interestingly, the loss of tuberin expression in human leiomyoma was associated with cytoplasmic p27Kip1 accumulation in this cell type. Furthermore, tuberin-null Eker rat leiomyomas and derived cell lines had predominantly cytoplasmic p27Kip1 compared to tuberin-expressing normal myometrium. Taken together, our data show that human and Eker rat leiomyoma proliferation is inhibited upon PPARγ treatment and that the etiology of human and Eker rat leiomyoma converge at loss of p27Kip1 function. Furthermore, our data indicate that the loss of p27 Kip1 function is mediated by loss of expression (in 80% of human leiomyoma) or cytoplasmic localization potentially resulting from the loss of tuberin. ^

Regulation of apoptosis during the pathogenesis of non -melanoma skin cancer

Previous studies from our lab have shown distinctive patterns of expression of bcl-2 gene family members in human nonmelanoma skin cancer (NMSC). To further evaluate the significance of these observations and to study the effects of cell death deregulation during skin carcinogenesis, we generated a transgenic mouse model (HK1.bcl-2) using the human keratin 1 promoter to target the expression of a human bcl-2 minigene to the epidermis. Transgenic protein expression was confirmed in all the layers of the epidermis except the stratum corneum using immunohistochemistry. Multifocal epidermal hyperplasia, without associated hyperkeratosis, was observed in newborn HK1.bcl-2 mice. Immunofluorescence staining using monoclonal antibodies specific for a variety of differentiation markers revealed aberrant expression of keratin 6 (K6) in the transgenic epidermis. Epidermal proliferative indexes, assessed by anti-BrdUrd immunofluorescence staining, were similar in control and transgenic newborn mice, but suprabasal proliferating cells were seen within the hyperplastic areas of the transgenic mouse skin. Spontaneous apoptotic indices of the epidermis were similar in both control and HK1.bcl-2 transgenic newborn mice, however, after UV-B irradiation, the number of "sunburn cells" was significantly higher in the control compared to the HK1.bcl-2 transgenic animals.^ Adult HK1.bcl-2 and control littermate mice were used in UV-B and chemical carcinogenesis protocols including DMBA + TPA. UV-B irradiated control and HK1.bcl-2 mice had comparable incidence of tumors than the controls, but the mean latency period was significantly shorter in the HK1.bcl-2 transgenic. Both control and transgenic animals included in chemical carcinogenesis protocols required application of both the initiating (DMBA) and promoting (TPA) agents to develop tumors. The frequency, number, and latency of tumor formation was similar in both groups of animals, however, HK1.bcl-2 mice exhibited a rate of conversion from benign papilloma to carcinoma 2.5 times greater than controls.^ Similar carcinogenesis experiments were performed using newborn mice. HK1.bcl-2 mice treated with UV-B plus TPA have a three fold greater incidence of tumor formation compared to controls littermates. HK1.bcl-2 transgenic animals also exhibited a shorter latency for papilloma formation when treated with DMBA plus TPA.^ HK1.bcl-2/v-Ha-ras double transgenic mice shared phenotypic features of both HK1.v-Ha-ras and HK1.bcl-2 transgenic mice, and exhibited focal areas of augmented hyperplasia. These double transgenic mice were susceptible to tumor formation by treatment with TPA alone.^ Cultures of primary keratinocytes were established from control, HK1.bcl-2, HK1.Ha-ras, and HK1.bcl-2/v-Ha-ras newborn mice. Cell viability was determined after exposure of the cells to UV-B irradiation, DMBA, TPA, or TGF-$\beta$1. Internucleosomal DNA fragmentation ("ladders") and morphological cellular changes compatible with apoptotic cell death were observed after the application of all these agents. HK1.bcl-2 keratinocytes were resistant to cell death induction by all of these agents except TGF-$\beta$1. HK1.Ha-ras cells had a higher spontaneous rate of cell death which could be compensated by co-expression of bcl-2.^ These findings suggest that bcl-2 dependent cell death suppression may be an important component of multistep skin carcinogenesis. ^

The role of erbB2 receptor in human breast cancer metastasis

Overexpression of c-erbB-2 gene-encoded p185 has been correlated with lymph node metastasis and poor prognosis in breast cancer patients. To investigate whether overexpression of c-erbB-2 can enhance metastatic potential of human breast cancer cells, we compared the metastatic phenotypes of the parental MDA-MB-435 cells and the c-erbB-2 gene transfected 435.eB cells. In vivo experimental metastasis assays demonstrated that mice injected erbB2-overexpressing 435.eB transfectants formed significantly more metastatic tumors than the mice injected with parental and control cells. The changes in metastatic potential in vivo were accompanied by increased invasiveness in vitro . The transfectants and the parental cells all had similar growth rates and transformation potential. These findings suggest that c- erbB-2 gene can enhance the intrinsic metastatic potentials of MDA-MB-435 cells without increasing their transformation abilities. ^ Homophilic adhesion may affect invasive and metastatic potential of tumor cells. We found that Heregulin-β1 (HRG-β1), a growth factor that activates receptor kinases erbB3 and erbB4, can enhance aggregation of MCF-7 and SKBR3 human breast cancer cells. While investigating the downstream signals involved in HRG-β1-increased cell aggregation, we observed that HRG-β1 increased the kinase activities of extracellular signal-regulated protein kinase (ERK) and PI3K in these cells. By using different kinase inhibitors, we found that the HRG-β1-activated MEK1-ERK pathway has no demonstrable role in the induction of cell aggregation, whereas HRG-β1-activated PI3K is required for enhancing breast cancer cell aggregation. These results have provided one mechanism by which HRG-β1-activated signaling of erbB receptors may affect invasive/metastatic properties of breast cancer cells. ^ To identify the structural motifs within the erbB2 receptor that are required for erbB2 increased metastatic potential in breast cancer cells, we injected different forms of mutated erbB2 expressing MDA-MB-435 cell line transfectants with or without the EGF-like domain of heregulin-β1 protein (HRG/egf) into ICR-SCID mice to test the metastatic survival rate. The results show that an intact kinase domain of erbB2 receptor is required for erbB2 enhanced metastatic potential in these cells. The C-terminal tyrosine 1248 residue of erbB2 may also play a role in enhancing metastatic potential. Moreover, the results suggest that HRG/egf promote the metastatic potential of human breast cancer cells in vivo. ^

Characterization and tissue-specific expression of the rat basic fibroblast growth factor antisense mRNA and protein

An RNA transcribed from the antisense strand of the FGF-2 gene has been implicated in the regulation of FGF-2 mRNA stability in amphibian oocytes. We have now cloned and characterized a novel 1.1-kb mRNA (fgf-as) from neonatal rat liver. In non-central nervous system (CNS) tissues the fgf-as RNA is abundantly expressed in a developmentally regulated manner. The FGF-AS cDNA contains a consensus polyadenylylation signal and a long open reading frame (ORF) whose deduced amino acid sequence predicts a 35-kDa protein with homology to the MutT family of nucleotide hydrolases. Western blot analysis with antibodies against the deduced peptide sequence demonstrates that the FGF-AS protein is expressed in a broad range of non-CNS tissue in the postnatal period. In the developing brain, the abundance of sense and antisense transcripts are inversely related, suggesting a role for the antisense RNA in posttranscriptional regulation of FGF-2 expression in this tissue.The FGF-AS is complementary to two widely separated regions in the long 3′ untranslated region of the FGF-2 mRNA, in the vicinity of the proximal and distal polyadenylylation sites. These findings demonstrate that the FGF-2 and fgf-as RNAs are coordinately transcribed on a tissue-specific and developmentally regulated basis and suggest that interaction of the sense and antisense RNAs may result in posttranscriptional regulation of FGF-2 in some tissues.

Cholesterol feeding reduces nuclear forms of sterol regulatory element binding proteins in hamster liver

Cholesterol feeding reduces the mRNAs encoding multiple enzymes in the cholesterol biosynthetic pathway and the low density lipoprotein receptor in livers of hamsters. Here we show that cholesterol feeding also reduces the levels of the nuclear NH2-terminal domains of sterol regulatory element binding proteins (SREBPs), which activate transcription of sterol-regulated genes. We show that livers of hamsters, like those of mice and humans, predominantly produce SREBP-2 and the 1c isoform of SREBP-1. Both are produced as membrane-bound precursors that must be proteolyzed to release the transcriptionally active NH2-terminal domains. Diets containing 0.1% to 1.0% cholesterol decreased the amount of nuclear SREBP-1c without affecting the amount of the membrane precursor or its mRNA, suggesting that cholesterol inhibits the proteolytic processing of SREBP-1 in liver as it does in cultured cells. Cholesterol also appeared to reduce the proteolytic processing of SREBP-2. In addition, at high levels of dietary cholesterol the mRNA encoding SREBP-2 declined and the amount of the precursor also fell, suggesting that cholesterol accumulation also may inhibit transcription of the SREBP-2 gene. The high-cholesterol diets reduced the amount of low density lipoprotein receptor mRNA by 30% and produced a more profound 70–90% reduction in mRNAs encoding 3-hydroxy-3-methylglutaryl CoA synthase and reductase. Treatment with lovastatin and Colestipol, which increases hepatic demands for cholesterol, increased the amount of SREBP-2 mRNA as well as the precursor and nuclear forms of the protein. This treatment caused a reciprocal decline in SREBP-1c mRNA and protein. Considered together, these data suggest that SREBPs play important roles in controlling transcription of sterol-regulated genes in liver, as they do in cultured cells.

The Gfi-1 protooncoprotein represses Bax expression and inhibits T-cell death

The Gfi-1 protooncogene encodes a nuclear zinc-finger protein that carries a novel repressor domain, SNAG, and functions as a position- and orientation-independent active transcriptional repressor. The Gfi-1 repressor allows interleukin 2 (IL-2)-dependent T cells to escape G1 arrest induced by IL-2 withdrawal in culture and collaborates with c-myc and pim-1 for the induction of retrovirus-induced lymphomas in animals. Here we show that overexpression of Gfi-1 also inhibits cell death induced by cultivation of IL-2-dependent T-cell lines in IL-2-deficient media. Similarly, induction of Gfi-1 in primary thymocytes from mice carrying a metal-inducible Gfi-1 transgene inhibits cell death induced by cultivation in vitro. The protein and mRNA levels of the proapoptotic regulator Bax are down-regulated by Gfi-1 in both immortalized T-cell lines and primary transgenic thymocytes. The repression is direct and depends on several Gfi-1-binding sites in the p53-inducible Bax promoter. In addition to Bax, Gfi-1 also represses Bak, another apoptosis-promoting member of the Bcl-2 gene family. Therefore, Gfi-1 may inhibit apoptosis by means of its repression of multiple proapoptotic regulators. The antiapoptotic properties of Gfi-1 provide a potential explanation for its strong collaboration with c-myc during oncogenesis.

Positive and negative regulation of endogenous genes by designed transcription factors

Gene regulation by imposed localization was studied by using designed zinc finger proteins that bind 18-bp DNA sequences in the 5′ untranslated regions of the protooncogenes erbB-2 and erbB-3. Transcription factors were generated by fusion of the DNA-binding proteins to repression or activation domains. When introduced into cells these transcription factors acted as dominant repressors or activators of, respectively, endogenous erbB-2 or erbB-3 gene expression. Significantly, imposed regulation of the two genes was highly specific, despite the fact that the transcription factor binding sites targeted in erbB-2 and erbB-3 share 15 of 18 nucleotides. Regulation of erbB-2 gene expression was observed in cells derived from several species that conserve the DNA target sequence. Repression of erbB-2 in SKBR3 breast cancer cells inhibited cell-cycle progression by inducing a G1 accumulation, suggesting the potential of designed transcription factors for cancer gene therapy. These results demonstrate the willful up- and down-regulation of endogenous genes, and provide an additional means to alter biological systems.

Circadian rhythms in Neurospora crassa: Lipid deficiencies restore robust rhythmicity to null frequency and white-collar mutants

The conidiation rhythm in the fungus Neurospora crassa is a model system for investigating the genetics of circadian clocks. Null mutants at the frq (frequency) locus (frq9 and frq10) make no functional frq gene products and are arrhythmic under standard conditions. The white-collar strains (wc-1 and wc-2) are insensitive to most effects of light, and are also arrhythmic. All three genes are proposed to be central components of the circadian oscillator. We have been investigating two mutants, cel (chain-elongation) and chol-1 (choline-requirer), which are defective in lipid synthesis and affect the period and temperature compensation of the rhythm. We have constructed the double mutant strains chol-1 frq9, chol-1 frq10, chol-1 wc-1, chol-1 wc-2, cel frq9, cel frq10, and cel wc-2. We find that these double mutant strains are robustly rhythmic when assayed under lipid-deficient conditions, indicating that free-running rhythmicity does not require the frq, wc-1, or wc-2 gene products. The rhythms in the double mutant strains are similar to the cel and chol-1 parents, except that they are less sensitive to light. This suggests that the frq, wc-1, and wc-2 gene products may be components of a pathway that normally supplies input to a core oscillator to transduce light signals and sustain rhythmicity. This pathway can be bypassed when lipid metabolism is altered.