Inhibition of p-mTOR represses transcription of PS1 and Notch 1-signaling.

Presenilin-1 (PS1) protein is the catalytic subunit of the gamma-secretase, and participates in the processing of beta-amyloid precursor protein (APP) to produce Abeta peptide and Notch 1 receptor to release Notch intracellular domain (NICD) in the cytoplasm. NICD migrates to the nucleus and causes Notch signaling by increasing the expression of the Hes1 gene. The mammalian target of rapamycin (mTOR) controls cellular homeostasis, and its activity is inhibited by rapamycin. The buildup of Abeta increases the mTOR signaling, whereas decreasing mTOR signaling reduces Abeta levels suggesting an interrelationship between mTOR signaling and Abeta. Administration of rapamycin in 3XTg-AD mouse model of Alzheimer's disease (AD) rescues cognitive deficits and ameliorates Abeta and Tau pathology. We have dissected the mechanisms by which rapamycin inhibits PS1 expression and Notch1 signaling. Our results demonstrated that rapamycin efficiently suppressed phosphorylation of mTOR (p-mTOR), and decreased expression of PS1-mRNA as well as p-p70S6K1, 4EBP1, PS1, NICD, and Hes1 protein levels. Therefore, rapamycin decreased PS1 protein levels and Notch 1 processing by inhibiting PS1 transcription.

Inhibition of p-mTOR represses PS1 transcription by reducing p-JNK.

Presenilin-1 (PS1) is the catalytic subunit of gamma-secretase. PS1 cleaves beta-amyloid precursor protein (APP) to produce Abeta peptide and Notch 1 receptor to release Notch intracellular domain (NICD) in the cytoplasm. We have previously shown that rapamycin inhibits p-mTOR to repress PS1 transcription and Notch 1-signaling. But the exact mechanism by which rapamycin inhibits PS1 transcription is not known. We have also published that inhibition of basal activity of c-jun-NH2-terminal kinase (JNK) with JNK-specific inhibitor SP600125 represses PS1 transcription by reducing p-JNK and via p53 dependent mechanism. We now report that rapamycin inhibits the phosphorylation of both mTOR (p-mTOR) and JNK (p-JNK). It appears that rapamycin represses PS1 transcription by inhibiting the expression of p-JNK in SK-N-SH cells under non-stressed condition. Consequently, one of the mechanisms of inhibition of PS1 transcription by rapamycin is similar to the mechanism of repression of PS1 transcription by JNK-specific inhibitor SP600125. We also report that JNK-inhibitor SP6000125 decreases both p-JNK and p-mTOR protein levels. These results suggest that JNK and mTOR may potentially activate each other by mutual phosphorylation.

HOXA5 Expression Is Elevated in Breast Cancer and Is Transcriptionally Regulated by Estradiol.

HOXA5 is a homeobox-containing gene associated with the development of the lung, gastrointestinal tract, and vertebrae. Here, we investigate potential roles and the gene regulatory mechanism in HOXA5 in breast cancer cells. Our studies demonstrate that HOXA5 expression is elevated in breast cancer tissues and in estrogen receptor (ER)-positive breast cancer cells. HOXA5 expression is critical for breast cancer cell viability. Biochemical studies show that estradiol (E2) regulates HOXA5 gene expression in cultured breast cancer cells in vitro. HOXA5 expression is also upregulated in vivo in the mammary tissues of ovariectomized female rats. E2-induced HOXA5 expression is coordinated by ERs. Knockdown of either ERα or ERß downregulated E2-induced HOXA5 expression. Additionally, ER co-regulators, including CBP/p300 (histone acetylases) and MLL-histone methylases (MLL2, MLL3), histone acetylation-, and H3K4 trimethylation levels are enriched at the HOXA5 promoter in present E2. In summary, our studies demonstrate that HOXA5 is overexpressed in breast cancer and is transcriptionally regulated via estradiol in breast cancer cells.

No Difference in Myosin Kinetics and Spatial Distribution of the Lever Arm in the Left and Right Ventricles of Human Hearts.

The systemic circulation offers larger resistance to the blood flow than the pulmonary system. Consequently, the left ventricle (LV) must pump blood with more force than the right ventricle (RV). The question arises whether the stronger pumping action of the LV is due to a more efficient action of left ventricular myosin, or whether it is due to the morphological differences between ventricles. Such a question cannot be answered by studying the entire ventricles or myocytes because any observed differences would be wiped out by averaging the information obtained from trillions of myosin molecules present in a ventricle or myocyte. We therefore searched for the differences between single myosin molecules of the LV and RV of failing hearts In-situ. We show that the parameters that define the mechanical characteristics of working myosin (kinetic rates and the distribution of spatial orientation of myosin lever arm) were the same in both ventricles. These results suggest that there is no difference in the way myosin interacts with thin filaments in myocytes of failing hearts, and suggests that the difference in pumping efficiencies are caused by interactions between muscle proteins other than myosin or that they are purely morphological.

The Role of Presenilin-1 in the Excitotoxicity of Ethanol Withdrawal.

Presenilin-1 (PS1) is a core component of γ-secretase that is involved in neurodegeneration. We have previously shown that PS1 interacts with a mitogen-activated protein kinase [(MAPK) jun-NH2-terminal-kinase], and another MAPK (p38) is activated by ethanol withdrawal (EW), abrupt termination from chronic ethanol exposure. EW is excitotoxic in nature, induces glutamate upregulation, and provokes neuronal damage. Here, we explored a potential mechanistic pathway involving glutamate, p38 (p38α isozyme), and PS1 that may mediate EW-induced excitotoxic stress. We used the prefrontal cortex of male rats withdrawn from a chronic ethanol diet. Additionally, we used ethanol-withdrawn HT22 cells (mouse hippocampal) treated with the inhibitor of glutamate receptors [dizocilpine (MK-801)], p38α (SB203580; 4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine), or γ-secretase [N-[N- (3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT)] during EW. Separately, ethanol-free HT22 cells were exposed to glutamate with or without SB203580 or DAPT. Protein levels, mRNA levels, and cell viability were assessed using immunoblotting, qualitative polymerase chain reaction, and calcein assay, respectively. The prefrontal cortex of ethanol-withdrawn rats or HT22 cells showed an increase in PS1 and p38α, which was attenuated by MK-801 and SB203580, but mimicked by glutamate treatment to ethanol-free HT22 cells. DAPT attenuated the toxic effect of EW or glutamate on HT22 cells. These results suggest that PS1 expression is triggered by glutamate through p38α, contributing to the excitotoxic stimulus of EW.

Membrane topology of human presenilin-1 in SK-N-SH cells determined by fluorescence correlation spectroscopy and fluorescent energy transfer.

Presenilin-1 (PS1) protein acts as passive ER Ca(2+) leak channels that facilitate passive Ca(2+) leak across ER membrane. Mutations in the gene encoding PS1 protein cause neurodegeneration in the brains of patients with familial Alzheimer's disease (FAD). FADPS1 mutations abrogate the function of ER Ca(2+) leak channel activity in human neuroblastoma SK-N-SH cells in vitro (Das et al., J Neurochem 122(3):487-500, 2012) and in mouse embryonic fibroblasts. Consequently, genetic deletion or mutations of the PS1 gene cause calcium (Ca(2+)) signaling abnormalities leading to neurodegeneration in FAD patients. By analogy with other known ion channels it has been proposed that the functional PS1 channels in ER may be multimers of several PS1 subunits. To test this hypothesis, we conjugated the human PS1 protein with an NH2-terminal YFP-tag and a COOH-terminal CFP-tag. As expected YFP-PS1, and PS1-CFP were found to be expressed on the plasma membranes by TIRF microscopy, and both these fusion proteins increased ER Ca(2+) leak channel activity similar to PS1 (WT) in SK-N-SH cells, as determined by functional calcium imaging. PS1-CFP was either expressed alone or together with YFP-PS1 into SK-N-SH cell line and the interaction between YFP-PS1 and PS1-CFP was determined by Förster resonance energy transfer analysis. Our results suggest interaction between YFP-PS1 and PS1-CFP confirming the presence of a dimeric or multimeric form of PS1 in SK-N-SH cells. Lateral diffusion of PS1-CFP and YFP-PS1 in the plasma membrane of SK-N-SH cells was measured in the absence or in the presence of glycerol by fluorescence correlation spectroscopy to show that both COOH-terminal and NH2-terminal of human PS1 are located on the cytoplasmic side of the plasma membrane. Therefore, we conclude that both COOH-terminal and NH2-terminal of human PS1 may also be oriented on the cytosolic side of ER membrane.

Repression of transcription of presenilin-1 inhibits γ-secretase independent ER Ca²âº leak that is impaired by FAD mutations.

Genetic deletion or mutations of presenilin genes (PS1/PS2) cause familial Alzheimer's disease and calcium (Ca²âº) signaling abnormalities. PS1/PS2 act as endoplasmic reticulum (ER) Ca²âº leak channels that facilitate passive Ca²âº leak across ER membrane. Studies with PS1/PS2 double knockout (PS1/PS2-DKO) mouse embryonic fibroblasts showed that PS1/PS2 were responsible for 80% of passive Ca²âº leak from the lumen of endoplasmic reticulum to cytosol. Transient transfection of the wild type PS1 expression construct increased cytoplasmic Ca²âº as a result of Ca²âº leak across ER membrane whereas the FADPS1 (PS1-M146V) mutation construct alone or in combination with the wild type PS1 expression construct abrogated Ca²âº leak in SK-N-SH cells. Inhibition of basal c-jun-NH2-terminal kinase (JNK) activity by JNK inhibitor SP600125 repressed PS1 transcription and PS1 protein expression by augmenting p53 protein level in SK-N-SH cells (Lee and Das 2008). In this report we also showed that repression of PS1 transcription by JNK inhibitor SP600125 inhibited passive Ca²âº leak across ER membrane which could be rescued by expressing PS1 wild type and not by expressing FADPS1 (PS1-M146V) under a SP600125 non-responsive promoter. Treatment of SK-N-SH cells with SP600125 also triggered InsP3R-mediated Ca²âº release from the ER by addition of 500 nM bradykinin, an agonist of InsP3 receptor (InsP3R1) without changing the expression of InsP3R1. This data confirms that SP600125 increases the Ca²âº store in the ER by inhibiting PS1-mediated Ca²âº leak across ER membrane. p53, ZNF237 and Chromodomain helicase DNA-binding protein 3 which are repressors of PS1 transcription, also reduced Ca²âº leak across ER membrane in SK-N-SH cells but γ-secretase inhibitor or dominant negative γ-secretase-specific PS1 mutant (PS1-D257A) had no significant effect. Therefore, p53, ZNF237, and Chromodomain helicase DNA-binding protein 3 inhibit the function ER Ca²âº leak channels to regulate both ER and cytoplasmic Ca²âº levels and may potentially control Ca²âº-signaling function of PS1.

Intraperitoneal injection of JNK-specific inhibitor SP600125 inhibits the expression of presenilin-1 and Notch signaling in mouse brain without induction of apoptosis.

Presenilin-1 (PS1) is a multifunctional protein involved in many cellular functions including the processing of type 1 membrane proteins such as ß-amyloid precursor protein (APP) and Notch 1 receptor. PS1 acts as the catalytic subunit of the γ-secretase complex, and participates in Notch 1 processing to release Notch intracellular domain (NICD) in the cytoplasm. NICD subsequently migrates to the nucleus and causes Notch signaling by increasing the expression of the Hes1 gene. We have previously shown that inhibition of basal activity of c-jun-NH2-terminal kinase (JNK) with JNK-specific inhibitor SP600125 represses the expression of PS1 and γ-secretase activity by increasing p53 level in SK-N-SH cell line in vitro (Lee and Das, 2008, 2010). However, it is largely unknown whether PS1 can be effectively suppressed in vivo in adult mouse brains. In this report we showed that intraperitoneal (i.p) injection of JNK-specific inhibitor SP600125 decreased p-JNK level, and reduced PS1 expression by increasing p53 level in adult mouse brains. We also showed that suppression of PS1 expression by SP600125 reduced γ-secretase activity which decreased Notch 1 processing to reduce NICD in mouse brains. Furthermore, inhibition of Notch 1 processing by SP600125 decreased Notch 1 signaling by reducing the expression of the NICD target Hes1 gene in mouse brains without induction of apoptosis. These results provide insights for further study on PS1-mediated reduction of Notch 1 and APP processing for the treatment of Alzheimer's disease.

Transcriptional regulation of the presenilin-1 gene controls gamma-secretase activity.

Inhibition of basal JNK activity by JNK inhibitor SP600125 or JNK1siRNA repressed presenilin-1 (PS1) expression in SK-N-SH cells by augmenting the level of p53, a repressor of the PS1 gene (1). We now showed that repression of PS1 transcription by JNK inhibitor SP600125 inhibited gamma-secretase mediated processing of amyloid precursor protein (APP) resulting in the accumulation of C99 fragment and the reduction of secreted Abeta40 level without altering the expression of nicastrin (NCT). Co-treatment of cells with SP600125 and p53 inhibitor, pifithrin-alpha, partially nullified the suppressive effects of SP610025 on PS1 expression and secreted Abeta40 level. Suppression of JNK1 by JNK1siRNA also decreased Abeta40 level. Furthermore, overexpression of the repressors p53, ZNF237 and CHD3 of the PS1 gene also suppressed the processing of APP through repression of PS1 transcription by deacetylation of histone at the PS1 promoter. Transcriptional activator Ets2 increased PS1 protein and secreted Abeta40 levels without affecting the expression of NCT by activating PS1 transcription via hyper-acetylation of histone at the PS1 promoter. Therefore, regulation of PS1 transcription modulates gamma-secretase activity.

Overexpression of human histone methylase MLL1 upon exposure to a food contaminant mycotoxin, deoxynivalenol.

Mixed lineage leukemias (MLLs) are histone-methylating enzymes with critical roles in gene expression, epigenetics and cancer. Although MLLs are important gene regulators little is known about their own regulation. Herein, to understand the effects of toxic stress on MLL gene regulation, we treated human cells with a common food contaminant mycotoxin, deoxynivalenol (DON). Our results demonstrate that MLLs and Hox genes are overexpressed upon exposure to DON. Studies using specific inhibitors demonstrated that Src kinase families are involved in upstream events in DON-mediated upregulation of MLL1. Sequence analysis demonstrated that the MLL1 promoter contains multiple Sp1-binding sites and importantly, the binding of Sp1 is enriched in the MLL1 promoter upon exposure to DON. Moreover, antisense-mediated knockdown of Sp1 diminished DON-induced MLL1 upregulation. These results demonstrated that MLL1 gene expression is sensitive to toxic stress and Sp1 plays crucial roles in the stress-induced upregulation of MLL1.

ADR1 interacts with a down-stream positive element to activate PS1 transcription.

We have identified downstream promoter sequence of the PS1 gene that may be regulated by novel transcription factors. 3' deletion from +178 to +165 had no effect on PS1 transcription. 3' deletion from +178 to +140 decreased promoter activity by 50%. Further 3' deletion from +178 to +114 decreased promoter activity by 80%. Therefore, a crucial element controlling over 80% of the promoter activity in SK-N-SH cell line is located between +114 and +165. Electrophoretic mobility shift assays suggested that zinc finger proteins Sp1 and ADR1 interacted with the PS1 promoter sequence (+114 to +140) and promoter region (+140 to +165) respectively. A three base pair substitution within the core sequence (GGCGGGGA to GGCGactA) of the ADR1 consensus in the element (+140 to +165) that abolished ADR1-DNA interaction, reduced PS1 transcription by 50%. The substitution mutation in the sequence (+114 to +140) that abolished Sp1-DNA interaction had no effect on PS1 expression. These data suggest that a novel mammalian trans-activator protein ADR1 binds to the downstream element (+140 to +165) to activate PS1 transcription.

Inhibition of basal activity of c-jun-NH2-terminal kinase (JNK) represses the expression of presenilin-1 by a p53-dependent mechanism.

Presenilin-1 (PS1) is a multifunctional protein involved in many cellular functions including the processing of type 1 transmembrane proteins and regulation of calcium signaling. Although PS1 is important in many aspects of cellular functions, little is known about the PS1 gene regulation in the context of intracellular signal pathways. We tested the role of c-jun-NH2-terminal kinase (JNK) on PS1 gene expression using a JNK specific inhibitor, SP600125. SP600125 efficiently suppressed basal JNK activity in SK-N-SH cell line as shown by inhibition of phosphor-JNK and phosphor-c-jun, and also decreased PS1 expression. Previously we reported that Ets1/2 bind to the PS1 promoter to activate PS1 transcription and p53 represses PS1 transcription without direct binding to the PS1 promoter [Pastorcic, M., Das, H.K., 2000. Regulation of transcription of the human presenilin-1 gene by ets transcription factors and the p53 protooncogene. J Biol Chem. 275, 34938-45.]. Involvement of protein-protein interaction between p53 and other transcription factors was speculated to be a mechanism by which p53 represses PS1 expression. Therefore, we tested whether the interaction between p53 and Ets1/2 is involved in JNK-mediated inhibition of PS1 expression. In this report we showed that p53 level was upregulated by SP600125 in SK-N-SH cell line. In addition, protein-protein interaction between p53 and Ets1/2 was enhanced with a concomitant dissociation of Ets1/2 from the PS1 promoter resulting in the suppression of PS1 transcription. We also showed that suppression of JNK1 by JNK1 siRNA increased p53 protein level and decreased PS1 expression. This observation was supported by the fact that overexpression of p53 in SK-N-SH cell line promoted dissociation of Ets1/2 from the PS1 promoter and suppressed PS1 expression. Furthermore, p53 inhibitor pifithrin-alpha partially nullified the suppressive effects of SP600125 on PS1 expression. We also showed that transfection of p53 was required for SP600125-mediated suppression of PS1 expression in p53-deficient PC3 cell line suggesting that inhibition of basal JNK activity suppresses PS1 expression through a p53-dependent mechanism.

Transcriptional regulation of the presenilin-1 gene: implication in Alzheimer's disease.

Two (amyloid and presenilin) hypotheses have been proposed to explain the pathogenesis of Alzheimer's disease (AD). According to amyloid hypothesis, the main amyloid plaques which are hallmark of AD are generated by beta- and gamma-secretase mediated proteolytic processing of amyloid precursor protein (APP). The amyloid hypothesis does not adequately address the pathogenesis of the disease, however, since transgenic mice that express the pathologic mutations of the APP and presenilin-1 (PS1) genes produce amyloid plaques but fail to exhibit neurodegeneration and memory loss observed in AD patients. According to presenilin hypothesis, loss of essential functions of PS due to decreased PS expression or mutations in the PS genes better explains the pathogenesis of AD. Recent studies have revealed that forebrain specific conditional knockouts of PS1 and PS2 genes (cPSKO) cause both neuronal degeneration and memory loss without evidence of formation of amyloid plaques. Another potential mechanism for the pathogenesis of AD may reside at the transcriptional regulation of the presenilin-1 gene. In this review, a detailed analysis of transcription factors that regulate PS1 transcription will be discussed. An in depth understanding of the regulatory mechanism of PS1 transcription can identify the targets that can potentially be used in therapeutic intervention of AD.

The C-terminal region of CHD3/ZFH interacts with the CIDD region of the Ets transcription factor ERM and represses transcription of the human presenilin 1 gene.

Presenilins are required for the function of gamma-secretase: a multiprotein complex implicated in the development of Alzheimer's disease (AD). We analyzed expression of the presenilin 1 (PS1) gene. We show that ERM recognizes avian erythroblastosis virus E26 oncogene homolog (Ets) motifs on the PS1 promoter located at -10, +90, +129 and +165, and activates PS1 transcription with promoter fragments containing or not the -10 Ets site. Using yeast two-hybrid selection we identified interactions between the chromatin remodeling factor CHD3/ZFH and the C-terminal 415 amino acids of ERM used as bait. Clones contained the C-terminal region of CHD3 starting from amino acid 1676. This C-terminal fragment (amino acids 1676-2000) repressed transcription of the PS1 gene in transfection assays and PS1 protein expression from the endogenous gene in SH-SY5Y cells. In cells transfected with both CHD3 and ERM, activation of PS1 transcription by ERM was eliminated with increasing levels of CHD3. Progressive N-terminal deletions of CHD3 fragment (amino acids 1676-2000) indicated that sequences crucial for repression of PS1 and interactions with ERM in yeast two-hybrid assays are located between amino acids 1862 and 1877. This was correlated by the effect of progressive C-terminal deletions of CHD3, which indicated that sequences required for repression of PS1 lie between amino acids 1955 and 1877. Similarly, deletion to amino acid 1889 eliminated binding in yeast two-hybrid assays. Testing various shorter fragments of ERM as bait indicated that the region essential for binding CHD3/ZFH is within the amino acid region 96-349, which contains the central inhibitory DNA-binding domain (CIDD) of ERM. N-Terminal deletions of ERM showed that residues between amino acids 200 and 343 are required for binding to CHD3 (1676-2000) and C-terminal deletions of ERM indicated that amino acids 279-299 are also required. Furthermore, data from chromatin immunoprecipitation (ChIP) indicate that CHD3/ZFH interacts with the PS1 promoter in vivo.

Analysis of transcriptional modulation of the presenilin 1 gene promoter by ZNF237, a candidate binding partner of the Ets transcription factor ERM.

DNA sequences required for the expression of the human presenilin 1 (PS1) gene have been identified between -118 and +178 flanking the major initiation site (+1) mapped in SK-N-SH cells. Several Ets sites are located both upstream as well as downstream from the +1 site, including an Ets motif present at -10 that controls 90% of transcription in SK-N-SH cells. However, in SH-SY5Y cells, transcription initiates further downstream and requires an alternative set of promoter elements including a +90 Ets motif. Ets2, ER81, ERM and Elk1 were identified by yeast one-hybrid selection in a human brain cDNA library using the -10 Ets motif as a bait. We have shown that ERM recognizes specifically Ets motifs on the PS1 promoter located at -10 as well as downstream at +90, +129 and +165 and activates PS1 transcription with promoter fragments whether or not they contain the -10 Ets site. We have now searched for ERM interacting proteins by yeast two-hybrid selection in a human brain cDNA library using the C-terminal 415 amino acid of ERM as a bait. One of the interacting proteins was ZNF237, a member of the MYM gene family. It is widely expressed in different tissues in eukaryotes under several forms derived by alternative splicing, including a large 382 amino acid form containing a single MYM domain, and 2 shorter forms of 208 and 213 amino acids respectively that do not. We show that both the 382 as well as the 208 amino acid forms are expressed in SK-N-SH cells but not in SH-SY5Y cells. Both forms interact with ERM and repress the transcription of PS1 in SH-SY5Y cells. The effect of both C-terminal and N-terminal deletions indicates that the N-terminal 120 amino acid region is required for interaction with ERM in yeast, and furthermore single amino acid mutations show that residues 112 and 114 play an important role. The repression of transcription in SH-SY5Y cells also appears to require the N-terminal potion of ZNF237 and was affected by mutation of the amino acid 112. Data from electrophoretic mobility shift assays indicate that ERM and possibly ZNF237 interact with a fragment of the PS1 promoter.

Alternative initiation of transcription of the human presenilin 1 gene in SH-SY5Y and SK-N-SH cells. The role of Ets factors in the regulation of presenilin 1.

We have identified DNA sequences required for the expression of the presenilin 1 (PS1) gene. A promoter region has been mapped in SK-N-SH cells and includes sequences between -118 and +178 flanking the major initiation site (+1). The PS1 gene is also efficiently transcribed in the SH-SY5Y subclone of SK-N-SH cells. However the promoter appears to be utilized in alternative ways in both cell types. Sequences both upstream as well as downstream from the initiation site mapped in SK-N-SH cells were shown by 5'- and 3'-deletion analysis to play a crucial role in both cell lines. However, in SH-SY5Y cells either upstream or downstream sequences are sufficient to direct transcription, whereas in SK-N-SH cells 5'-deletions past the +1 site eliminate over 95% of transcription. Several Ets motifs (GGAA) as well as Sp1 motifs [(G/T)GGCGGRRY] are juxtaposed both upstream and downstream from +1. To understand how the promoter may be utilized alternatively in different cell types we have examined the effect of point mutations in these elements. Altering an Ets motif at -10 eliminates 80% of transcription in SK-N-SH cells whereas the same mutation has only a minor effect in SH-SY5Y cells. Conversely, mutation of the Ets element at +90, which eliminates 70% of transcription in SH-SY5Y cells, has a lesser effect in SK-N-SH cells. In both cell types a promoter including mutations at both -10 and +90 sites loses over 90% transcription activity indicating the crucial importance of these two Ets motifs. The effect of Sp1 mutations appears to be similar in both cell types. Hence the differential expression in each cell type may be at least partially determined by Ets factors and the -10/+90 sites. We have identified several Ets factors that recognize specifically the -10 Ets motif by the yeast one-hybrid selection including avian erythroblastosis virus E26 oncogene homologue 2, Ets-like gene 1, Ets translocation variant 1 and Ets related molecule (ERM). We show here that ERM specifically recognizes Ets motifs on the PS1 promoter located at -10 as well as downstream at +90, +129 and +165 and activates PS1 transcription with promoter fragments containing or not the -10 Ets site.

Regulatory roles of presenilin-1 and nicastrin in neuronal differentiation during in vitro neurogenesis.

Presenilin (PS) in association with nicastrin (NICA) forms a gamma-secretase complex that plays a crucial role in facilitating intramembranous processing of Notch, a signaling receptor that is essential for neuronal fate specification and differentiation. Loss of function studies have implicated a role for PS1 in regulating neuronal differentiation in association with the down-regulation of Notch signaling during neurogenesis. By using a system for stable, as well as tetracycline-inducible expression of interfering RNAs (RNAi), we studied the functions of PS1 during neuronal differentiation in the murine pluripotent p19 embryonic carcinoma cell line. After retinoic acid (RA) treatment and in the absence of doxycycline, neuronal progenitor cells in the p19 clone were found to extend their processes towards the neighboring colony to form network-like connections, as revealed by neuron-specific microtubule-associated protein 2 staining and laser scanning confocal microscopy. However, doxycycline-induced expression of PS1 small interfering RNA (siRNA) in the p19 clone resulted in a severe defect in the formation of network-like connections. Expression of the NICA and Notch down-stream effector genes Hes1 and Hes5 was unaffected in p19 cells expressing doxycycline-induced PS1 siRNA. In contrast to PS1, constitutive inactivation of NICA by siRNA in p19 cells resulted in premature and partial differentiation without RA treatment. In these NICA siRNA-expressing p19 cells the expression of the Notch1 down-stream effector Hes1 gene was substantially reduced. After RA treatment the NICA siRNA clone failed to differentiate completely into networks of neurons. These results taken together provide direct evidence that PS1 and NICA may participate in neuronal differentiation during neurogenesis in vitro.

An abbreviated procedure for the cloning and identification of Ets transcription factors regulating the expression of the human presenilin 1 gene.

We have previously defined a crucial DNA element controlling 90% of the expression of the presenilin 1 gene at (-35 to +6). This region contains an Ets transcription factor binding motif, and a two-basepair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by over 90%. We have shown that Ets1/2 transcription factors bind specifically to the -10 Ets element and activate PS1 transcription. The identification of other transcription factors recognizing specifically this promoter area should provide insights into the regulation of PS1. We have used the -10 Ets element as a bait in yeast one hybrid screening of a human brain cDNA library using a His3 reporter construct. We describe an abbreviated one-hybrid protocol to screen cDNA libraries. This assay selected four factors from the Ets family: Ets2, ER81, ERM and Elk1. We have also shown that specific DNA binding activity to the -10 Ets element of PS1 could easily be detected in yeast clones by EMSAs including protein extracts from yeast cells, thus confirming specific DNA binding activity without further sequencing and subcloning into suitable expression vectors. Ultimately the identity of putative clones was confirmed by DNA sequencing. We also confirmed the specific DNA binding properties of the factors identified by showing that the proteins produced by in vitro translation of the entire cDNAs from Elk1 and ER81 indeed binds specifically to the -10 region of the PS1 promoter.

Ets transcription factors ER81 and Elk1 regulate the transcription of the human presenilin 1 gene promoter.

We have previously defined a crucial DNA element controlling 90% of the expression of the presenilin 1 gene at (-35 to +6). This region contains an Ets transcription factor binding motif, and a 2-base pair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by over 90%. We have shown that Ets1/2 transcription factors bind specifically to the -10 Ets element and activate PS1 transcription. The identification of other transcription factors recognizing specifically this promoter area should provide insights into the regulation of PS1. We have used the -10 Ets element as a bait in yeast one hybrid screening of a human brain cDNA library. This assay selected three factors from the Ets family: Ets2, ER81 and Elk1. We show that in vitro translated ER81 indeed binds specifically to the -10 region of the PS1 promoter and that ER81 activates by two- to threefold the basal transcription of a presenilin-1 promoter-chloramphenicol acetyltransferase reporter synthetic gene (-119, +178)PS1CAT in transient infection assays in neuroblastoma cells (SK-N-SH). GABPalpha, a member of the Ets family closely related to Ets2 and also containing a pointed domain, only increased PS1 transcription by about twofold. Cotransfection of GABPbeta together with GABPalpha did not increase PS1 transcription. However, GABPbeta alone activated PS1 transcription by two- to threefold. In contrast, the more distantly related Ets factor Elk1 repressed PS1 transcription very effectively.

Activation of transcription of the human presenilin 1 gene by 12-O-tetradecanoylphorbol 13-acetate.

We have recently identified an Ets element controlling over 90% of the basal expression of the human presenilin 1 (PS1) gene. We have also shown that Ets1 and Ets2 act as transactivators of the PS1 gene by cotransfection experiments in SK-N-SH neuronal cells. The PS1 gene is widely but differentially expressed across tissues and the expression in brain appears to be restricted to neurons. To gain further insight into the regulation of the gene we have examined the regulation of PS1 by 12-O-tetradecanoylphorbol 13-acetate (TPA). SK-N-SH neuronal cells were treated with 0.2 micro m TPA for 30 min to 24 h and the level of expression of the endogenous PS1 gene was measured by Northern blot analysis. A two- to threefold increase in the level of PS1 mRNA appeared 4-8 h after the addition of TPA. A similar increase in transcription activity was observed in nuclear run off experiments, indicating that the increased mRNA level results from an activation in the initiation of transcription of PS1. Consistently, TPA also increased the level of PS1 protein. No activation of the PS1 endogenous gene by TPA was observed in hepatoma HepG2 cells. Next we tested the effect of TPA on the expression of the PS1 promoter by transfecting fusion genes including various fragments of the PS1 promoter linked to a CAT reporter into SK-N-SH cells. TPA also stimulated the expression of the PS1CAT constructs. Generally wild type constructs -687/+178, -118/+178, -22/+178 including the short -35/+6 fragment showed a minor two- to threefold activation by TPA. Point mutations eliminating the -10 Ets motif or the -6 CREB/AP1 motif did not decrease the stimulation by TPA. Thus TPA appears to activate the transcription of the PS1 gene by a mechanism which does not require these elements.