Hriday K. Das

Regulation of Transcription of the Human Presenilin-1 Gene by Ets Transcription Factors and the p53 Protooncogene

The expression of the human presenilin-1 cellular gene is suppressed by the p53 protooncogene. The rapid kinetic of the down-regulation has suggested that it may result from a primary mechanism. We show here that p53 also suppresses the transcription of a presenilin-1 promoter-chloramphenicol acetyltransferase reporter synthetic gene in transient infection assays in neuroblastoma (SK-N-SH) and hepatoma (HepG2) cell lines. Only a minimum promoter including sequences from −35 to + 6 from the transcription initiation is sufficient to confer down-regulation. We have previously defined a crucial DNA element controlling 90% of the expression of the gene within the same short area, and the identification of the transcription factors involved should also provide insights into the regulation ofPS1 by p53. 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 more than 90%. We now show that Ets1 and Ets2 indeed transactivate a PS1 promoter-chloramphenicol acetyltransferase reporter including the (−35 to +6) fragment. Furthermore, in vitro translated Ets2 binds specifically to the −10 Ets motif in electrophoretic mobility shift assays. Therefore, Ets1/2 factors bind specifically to the −10 Ets element and activatePS1 transcription. We also show that the coactivator p300 enhances the activation by Ets1 and Ets2 as well as the repression by p53. p300 is known to interact with p53 as well as with Ets1 and Ets2. We show that p53 does not bind directly to the PS1promoter. Hence the repression of PS1 transcription by p53 is likely to be mediated through protein-protein interactions.

An Upstream Element Containing an ETS Binding Site Is Crucial for Transcription of the Human Presenilin-1 Gene

Deletion mapping of the human presenilin-1 (PS1) promoter delineated the most active fragment from −118 to +178 in relation to the transcription start site mapped in this study, in both human neuroblastoma SK-N-SH and hepatoma HepG2 cells. 5′ deletions revealed that a crucial element controlling over 90% of the promoter activity in these cell lines is located between −22 and −6. A mutation altering only two nucleotides of the ETS consensus sequence present at −12 (GGAA to TTAA) has a similar effect. Electrophoretic mobility shift assays showed that a set of specific complexes between nuclear factors and the PS1 promoter are eliminated by this point mutation, as well as by competition with an ETS consensus oligonucleotide. Competition experiments in DNase I footprinting correlated with electrophoretic mobility shift assays and showed that only one of several footprints over the PS1 promoter is eliminated by competition with an ETS consensus oligonucleotide. It extends from −14 to −6 and surrounds the ETS motif present at −12. Thus, a crucial ETS element is present at −12 and binds a protein(s) recognizing specifically the ETS consensus motif. At least one such complex is eliminated by preincubating the nuclear extract with an antibody with broad cross-reactivity with Ets-1 and Ets-2 proteins, thus confirming that an ETS transcription factor(s) recognizes the −12 motif. Several Sp1 binding motifs at positions −70, −55, and +20 surround this ETS element. Competition DNase I footprinting showed that Sp1-like nuclear factors recognize specifically these sites in both cell lines. Furthermore, a combination of 5′ and 3′ deletions indicated the presence of positive promoter elements between −96 and −35 as well as between +6 and +42. Thus, transfection and footprinting assays correlate to suggest that Sp1 transcription factor(s) bind at several sites upstream and downstream from the initiation site and activate the transcription of the PS1 promoter. Sequences downstream from the transcription initiation site also contain major control elements. 3′ deletions from +178 to +107 decreased promoter activity by 80%. However, further deletion to +42 increased promoter activity by 3–4-fold. Collectively, these data indicate that sequences upstream and downstream from the transcription start site each control over 80% of the promoter activity. Hence, this suggests that protein-protein interactions between factors recognizing downstream and upstream sequences are involved.

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.

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.

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

Presenilin (PS) in association with nicastrin (NICA) forms a γ‐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.

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.

Repression of transcription of presenilin‐1 inhibits γ‐secretase independent ER Ca2+ leak that is impaired by FAD mutations

J. Neurochem. (2012) 122, 487–500.

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 β-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 Alzheimers disease.

Evidence for Pre- and Post-Power Stroke of Cross-Bridges of Contracting Skeletal Myofibrils

We examined the orientational fluctuations of a small number of myosin molecules (approximately three) in working skeletal muscle myofibrils. Myosin light chain 1 (LC1) was labeled with a fluorescent dye and exchanged with the native LC1 of skeletal muscle myofibrils cross-linked with 1-ethyl-3-[3(dimethylamino) propyl] carbodiimide to prevent shortening. We observed a small volume within the A-band (∼10(-15) L) by confocal microscopy, and measured cyclic fluctuations in the orientation of the myosin neck (containing LC1) by recording the parallel and perpendicular components of fluorescent light emitted by the fluorescently labeled myosin LC1. Histograms of orientational fluctuations from fluorescent molecules in rigor were represented by a single Gaussian distribution. In contrast, histograms from contracting muscles were best fit by at least two Gaussians. These results provide direct evidence that cross-bridges in working skeletal muscle assume two distinct conformations, presumably corresponding to the pre- and post-power-stroke states.

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.