Chromatin architecture at susceptible gene loci in cerebellar Purkinje cells characterizes DNA damage-induced neurodegeneration.

The pathogenesis of inherited genome instability neurodegenerative syndromes remains largely unknown. Here, we report new disease-relevant murine models of genome instability­driven neurodegeneration involving disabled ATM and APTX that develop debilitating ataxia. We show that neurodegeneration and ataxia result from transcriptional interference in the cerebellum via aberrant messenger RNA splicing. Unexpectedly, these splicing defects were restricted to only Purkinje cells, disrupting the expression of critical homeostatic regulators including ITPR1, GRID2, and CA8. Abundant genotoxic R loops were also found at these Purkinje cell gene loci, further exacerbating DNA damage and transcriptional disruption. Using ATAC-seq to profile global chromatin accessibility in the cerebellum, we found a notably unique chromatin conformation specifically in Purkinje chromatin at the affected gene loci, thereby promoting susceptibility to DNA damage. These data reveal the pathogenic basis of DNA damage in the nervous system and suggest chromatin conformation as a feature in directing genome instability­associated neuropathology.

Genome instability independent of type I interferon signaling drives neuropathology caused by impaired ribonucleotide excision repair.

Aicardi-Goutières syndrome (AGS) is a monogenic type I interferonopathy characterized by neurodevelopmental defects and upregulation of type I interferon signaling and neuroinflammation. Mutations in genes that function in nucleic acid metabolism, including RNASEH2, are linked to AGS. Ribonuclease H2 (RNASEH2) is a genome surveillance factor critical for DNA integrity by removing ribonucleotides incorporated into replicating DNA. Here we show that RNASEH2 is necessary for neurogenesis and to avoid activation of interferon-responsive genes and neuroinflammation. Cerebellar defects after RNASEH2B inactivation are rescued by p53 but not cGAS deletion, suggesting that DNA damage signaling, not neuroinflammation, accounts for neuropathology. Coincident inactivation of Atm and Rnaseh2 further affected cerebellar development causing ataxia, which was dependent upon aberrant activation of non-homologous end-joining (NHEJ). The loss of ATM also markedly exacerbates cGAS-dependent type I interferon signaling. Thus, DNA damage-dependent signaling rather than type I interferon signaling underlies neurodegeneration in this class of neurodevelopmental/neuroinflammatory disease.

Long-term nationwide assessment of polychlorinated dibenzo-p-dioxins/dibenzofurans and dioxin-like polychlorinated biphenyls ambient air concentrations for ten years in South Korea.

In this study, seasonal/regional variations of Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/DFs) and dioxin like-polychlorinated biphenyls in the ambient air were monitored for ten years (2008-2017) using a high volume air sampler. As a result of strict regulation enforced by Korea Ministry of Environment in 2008, PCDD/DFs concentrations in the ambient air decreased from 0.051 pg I-TEQ Sm-3 in 2009 to 0.014 pg I-TEQ Sm-3 in 2017 which was comparably associated with cut-down of their emission sources from 880.2 g I-TEQ Sm-3 in 2001 to 24.2 g I-TEQ Sm-3 in 2015; revealing that it was only 2.7% against that of 2001. In 2017, mean TEQ concentration level of PCDD/DFs in the air of South Korea was quite low in comparison to its ambient environmental standards of 0.6 pg I-TEQ Sm-3 for PCDD/DFs. Particularly, the sum of PCDD/DFs in the background revealed the lowest level, however, the fraction of octachlorodibenzodioxin among other isomers exposed at the highest level in this study, suggesting that the ambient air quality in the background being studied was severely and persistently impaired by inflowing unknown sources of any possible anthropogenic transboundary migratory air pollutants. Moreover, this study conducted the scientific analysis of the long-term variations in the ambient air and emission sources using principal component analysis. From this of 10 years long-term nationwide assessments for the PCDD/DFs and dl-PCBs in the ambient air, it is possible to prove that South Korean environmental policy to manage POPs has been successfully conducted for the last ten years.

Apurinic endonuclease-1 preserves neural genome integrity to maintain homeostasis and thermoregulation and prevent brain tumors.

Frequent oxidative modification of the neural genome is a by-product of the high oxygen consumption of the nervous system. Rapid correction of oxidative DNA lesions is essential, as genome stability is a paramount determinant of neural homeostasis. Apurinic/apyrimidinic endonuclease 1 (APE1; also known as "APEX1" or "REF1") is a key enzyme for the repair of oxidative DNA damage, although the specific role(s) for this enzyme in the development and maintenance of the nervous system is largely unknown. Here, using conditional inactivation of murine Ape1, we identify critical roles for this protein in the brain selectively after birth, coinciding with tissue oxygenation shifting from a placental supply to respiration. While mice lacking APE1 throughout neurogenesis were viable with little discernible phenotype at birth, rapid and pronounced brain-wide degenerative changes associated with DNA damage were observed immediately after birth leading to early death. Unexpectedly, Ape1Nes-cre mice appeared hypothermic with persistent shivering associated with the loss of thermoregulatory serotonergic neurons. We found that APE1 is critical for the selective regulation of Fos1-induced hippocampal immediate early gene expression. Finally, loss of APE1 in combination with p53 inactivation resulted in a profound susceptibility to brain tumors, including medulloblastoma and glioblastoma, implicating oxidative DNA lesions as an etiologic agent in these diseases. Our study reveals APE1 as a major suppressor of deleterious oxidative DNA damage and uncovers specific and broad pathogenic consequences of respiratory oxygenation in the postnatal nervous system.

The Hippo Pathway Prevents YAP/TAZ-Driven Hypertranscription and Controls Neural Progenitor Number.

The Hippo pathway controls the activity of YAP/TAZ transcriptional coactivators through a kinase cascade. Despite the critical role of this pathway in tissue growth and tumorigenesis, it remains unclear how YAP/TAZ-mediated transcription drives proliferation. By analyzing the effects of inactivating LATS1/2 kinases, the direct upstream inhibitors of YAP/TAZ, on mouse brain development and applying cell-number-normalized transcriptome analyses, we discovered that YAP/TAZ activation causes a global increase in transcription activity, known as hypertranscription, and upregulates many genes associated with cell growth and proliferation. In contrast, conventional read-depth-normalized RNA-sequencing analysis failed to detect the scope of the transcriptome shift and missed most relevant gene ontologies. Following a transient increase in proliferation, however, hypertranscription in neural progenitors triggers replication stress, DNA damage, and p53 activation, resulting in massive apoptosis. Our findings reveal a significant impact of YAP/TAZ activation on global transcription activity and have important implications for understanding YAP/TAZ function.

Secreted type of amyloid precursor protein induces glial differentiation by stimulating the BMP/Smad signaling pathway.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases leading to dementia. Although cytotoxicity of amyloid ß peptides has been intensively studied within pathophysiology of AD, the physiological function of amyloid precursor protein (APP) still remains unclarified. We have shown previously that secreted APPα (sAPPα) is associated with glial differentiation of neural stem cells. To elucidate specific mechanisms underlying sAPPα-induced gliogenesis, we examined the potential involvement of bone morphogenic proteins (BMPs). BMPs are one of the factors involved in glial differentiation of neural progenitor cells. When expressions of BMP-2, -4, and -7 were examined, upregulation of BMP-4 expression was solely observed as a result of treatment with sAPPα in a time and dose-dependent manner. Furthermore, the treatment of sAPPα promoted phosphorylation of Smad1/5/8, a downstream signaling mediator of BMP receptors. Interestingly, N-terminal domain of APP (1-205) was sufficient to elevate BMP4 expression, resulting in an increase of glial fibrillary acidic protein (GFAP) expression and phosphorylation of Smad1/5/8. However, the application of APP neutralizing antibody and anti-BMP4 antibody significantly suppressed expression of BMP-4 as well as phosphorylation of Smad1/5/8. Thus, our results indicate that sAPPα-induced gliogenesis is in part mediated by the BMP-4 signaling pathway. We also observed upregulation of BMP-4 and phosphorylation of Smad1/5/8 in APP transgenic mice. It is imperative to unravel the mechanisms underlying the role of BMP-4 during APPα-induced glial differentiation in hope of providing novel prevention or treatment for AD.

Metabolic stress modulates Alzheimer's ß-secretase gene transcription via SIRT1-PPARγ-PGC-1 in neurons.

Classic cardio-metabolic risk factors such as hypertension, stroke, diabetes, and hypercholesterolemia all increase the risk of Alzheimer's disease. We found increased transcription of ß-secretase/BACE1, the rate-limiting enzyme for Aß generation, in eNOS-deficient mouse brains and after feeding mice a high-fat, high-cholesterol diet. Up- or downregulation of PGC-1α reciprocally regulated BACE1 in vitro and in vivo. Modest fasting in mice reduced BACE1 transcription in the brains, which was accompanied by elevated PGC-1 expression and activity. Moreover, the suppressive effect of PGC-1 was dependent on activated PPARγ, likely via SIRT1-mediated deacetylation in a ligand-independent manner. The BACE1 promoter contains multiple PPAR-RXR sites, and direct interactions among SIRT1-PPARγ-PGC-1 at these sites were enhanced with fasting. The interference on the BACE1 gene identified here represents a unique noncanonical mechanism of PPARγ-PGC-1 in transcriptional repression in neurons in response to metabolic signals that may involve recruitment of corepressor NCoR.

Upregulation of the E3 ligase NEDD4-1 by oxidative stress degrades IGF-1 receptor protein in neurodegeneration.

The importance of ubiquitin E3 ligases in neurodegeneration is being increasingly recognized. The crucial role of NEDD4-1 in neural development is well appreciated; however, its role in neurodegeneration remains unexplored. Herein, we report increased NEDD4-1 expression in the degenerated tissues of several major neurodegenerative diseases. Moreover, its expression is upregulated in cultured neurons in response to various neurotoxins, including zinc and hydrogen superoxide, via transcriptional activation likely mediated by the reactive oxygen species (ROS)-responsive FOXM1B. Reduced protein levels of the insulin-like growth factor receptor (IGF-1Rß) were observed as a consequence of upregulated NEDD4-1 via the ubiquitin-proteasome system. Overexpression of a familial mutant form of superoxide dismutase 1 (SOD1) (G93A) in neuroblastoma cells resulted in a similar reduction of IGF-1Rß protein. This inverse correlation between NEDD4-1 and IGF-1Rß was also observed in the cortex and spinal cords of mutant (G93A) SOD1 transgenic mice at a presymptomatic age, which was similarly induced by in vivo-administered zinc in wild-type C57BL/6 mice. Furthermore, histochemistry reveals markedly increased NEDD4-1 immunoreactivity in the degenerating/degenerated motor neurons in the lumbar anterior horn of the spinal cord, suggesting a direct causative role for NEDD4-1 in neurodegeneration. Indeed, downregulation of NEDD4-1 by shRNA or overexpression of a catalytically inactive form rescued neurons from zinc-induced cell death. Similarly, neurons with a NEDD4-1 haplotype are more resistant to apoptosis, largely due to expression of higher levels of IGF-1Rß.Together, our work identifies a novel molecular mechanism for ROS-upregulated NEDD4-1 and the subsequently reduced IGF-1Rß signaling in neurodegeneration.

Differential regulation of BACE1 expression by oxidative and nitrosative signals.

BACKGROUND: It is well established that both cerebral hypoperfusion/stroke and type 2 diabetes are risk factors for Alzheimer's disease (AD). Recently, the molecular link between ischemia/hypoxia and amyloid precursor protein (APP) processing has begun to be established. However, the role of the key common denominator, namely nitric oxide (NO), in AD is largely unknown. In this study, we investigated redox regulation of BACE1, the rate-limiting enzyme responsible for the ß-cleavage of APP to Aß peptides. RESULTS: Herein, we studied events such as S-nitrosylation, a covalent modification of cysteine residues by NO, and H2O2-mediated oxidation. We found that NO and H2O2 differentially modulate BACE1 expression and enzymatic activity: NO at low concentrations (<100 nM) suppresses BACE1 transcription as well as its enzymatic activity while at higher levels (0.1-100 µM) NO induces S-nitrosylation of BACE1 which inactivates the enzyme without altering its expression. Moreover, the suppressive effect on BACE1 transcription is mediated by the NO/cGMP-PKG signaling, likely through activated PGC-1α. H2O2 (1-10 µM) induces BACE1 expression via transcriptional activation, resulting in increased enzymatic activity. The differential effects of NO and H2O2 on BACE1 expression and activity are also reflected in their opposing effects on Aß generation in cultured neurons in a dose-dependent manner. Furthermore, we found that BACE1 is highly S-nitrosylated in normal aging brains while S-nitrosylation is markedly reduced in AD brains. CONCLUSION: This study demonstrates for the first time that BACE1 is highly modified by NO via multiple mechanisms: low and high levels of NO suppress BACE1 via transcriptional and post translational regulation, in contrast with the upregulation of BACE1 by H2O2-mediated oxidation. These novel NO-mediated regulatory mechanisms likely protect BACE1 from being further oxidized by excessive oxidative stress, as from H2O2 and peroxynitrite which are known to upregulate BACE1 and activate the enzyme, resulting in excessive cleavage of APP and Aß generation; they likely represent the crucial house-keeping mechanism for BACE1 expression/activation under physiological conditions.

Involvement of notch signaling pathway in amyloid precursor protein induced glial differentiation.

The amyloid precursor protein (APP) has been mainly studied in its role in the production of amyloid ß peptides (Aß), because Aß deposition is a hallmark of Alzheimer's disease. Although several studies suggest APP has physiological functions, it is still controversial. We previously reported that APP increased glial differentiation of neural progenitor cells (NPCs). In the current study, NPCs transplanted into APP23 transgenic mice primarily differentiated into glial cells. In vitro treatment with secreted APP (sAPP) dose-dependently increased glial fibrillary acidic protein (GFAP) immuno-positive cells in NPCs and over expression of APP caused most NPCs to differentiate into GFAP immuno-positive cells. Treatment with sAPP also dose-dependently increased expression levels of GFAP in NT-2/D1 cells along with the generation of Notch intracellular domain (NICD) and expression of Hairy and enhancer of split 1 (Hes1). Treatment with γ-secretase inhibitor suppressed the generation of NICD and reduced Hes1 and GFAP expressions. Treatment with the N-terminal domain of APP (APP 1-205) was enough to induce up regulation of GFAP and Hes1 expressions, and application of 22 C11 antibodies recognizing N-terminal APP suppressed these changes by sAPP. These results indicate APP induces glial differentiation of NPCs through Notch signaling.

NO signaling and S-nitrosylation regulate PTEN inhibition in neurodegeneration.

BACKGROUND: The phosphatase PTEN governs the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway which is arguably the most important pro-survival pathway in neurons. Recently, PTEN has also been implicated in multiple important CNS functions such as neuronal differentiation, plasticity, injury and drug addiction. It has been reported that loss of PTEN protein, accompanied by Akt activation, occurs under excitotoxic conditions (stroke) as well as in Alzheimer's (AD) brains. However the molecular signals and mechanism underlying PTEN loss are unknown. RESULTS: In this study, we investigated redox regulation of PTEN, namely S-nitrosylation, a covalent modification of cysteine residues by nitric oxide (NO), and H2O2-mediated oxidation. We found that S-nitrosylation of PTEN was markedly elevated in brains in the early stages of AD (MCI). Surprisingly, there was no increase in the H2O2-mediated oxidation of PTEN, a modification common in cancer cell types, in the MCI/AD brains as compared to normal aged control. Using several cultured neuronal models, we further demonstrate that S-nitrosylation, in conjunction with NO-mediated enhanced ubiquitination, regulates both the lipid phosphatase activity and protein stability of PTEN. S-nitrosylation and oxidation occur on overlapping and distinct Cys residues of PTEN. The NO signal induces PTEN protein degradation via the ubiquitin-proteasome system (UPS) through NEDD4-1-mediated ubiquitination. CONCLUSION: This study demonstrates for the first time that NO-mediated redox regulation is the mechanism of PTEN protein degradation, which is distinguished from the H2O2-mediated PTEN oxidation, known to only inactivate the enzyme. This novel regulatory mechanism likely accounts for the PTEN loss observed in neurodegeneration such as in AD, in which NO plays a critical pathophysiological role.

Amyloid-ß precursor protein induces glial differentiation of neural progenitor cells by activation of the IL-6/gp130 signaling pathway.

Although amyloid precursor protein (APP) due to the cytotoxicity of Aß peptides, has been intensively studied, the physiological role of APP still remains wrapped up in veil. In this article, we propose that α-cleaved ectodomain of APP (sAPPα) stimulates the IL-6/gp130 signaling pathway for induction of gliogenesis within neural progenitor cells (NPCs). In our previous study, a high dose of APP differentiated NPCs into glial fibrillary acidic protein (GFAP) positive cells. In order to elucidate the mechanism of APP-induced glial differentiation, we examined the effects of sAPPα on the IL-6/gp130 signaling pathway. Application of sAPPα promoted mRNA expression of gp130, ciliary neurotrophic factor (CNTF), and Janus kinase 1 (JAK1). sAPPα stimulated the glial differentiation by upregulating the expression and phosphorylation of gp130. While mRNA expression of STAT3 was unchanged, phosphorylation of STAT3-Tyr705 gradually increased. Application of small interference RNA (siRNA) for STAT3 suppressed GFAP expression even in the presence of APP. Treatment with siRNA or inhibitor, AG490, of JAK1 efficiently suppressed STAT3 phosphorylation and GFAP expression. Upregulation of CNTF was observed in either short- or long-term treatment with sAPPα. RNA's interference of CNTF dose-dependently inhibited GFAP expression upregulated by treatment with sAPPα. This study suggests that the IL-6/gp130 signaling pathway is involved in sAPPα-induced glial differentiation of NPCs. Although further investigation is needed, this study may provide insight into the mechanism of glial differentiation of NPCs under pathological conditions in Alzheimer's disease or Down syndrome.

Functional interaction of phosphatase and tensin homologue (PTEN) with the E3 ligase NEDD4-1 during neuronal response to zinc.

The contribution of zinc-mediated neuronal death in the process of both acute and chronic neurodegeneration has been increasingly appreciated. Phosphatase and tensin homologue, deleted on chromosome 10 (PTEN), the major tumor suppressor and key regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, plays a critical role in neuronal death in response to various insults. NEDD4-1-mediated PTEN ubiquitination and subsequent degradation via the ubiquitin proteosomal system have recently been demonstrated to be the important regulatory mechanism for PTEN in several cancer types. We now demonstrate that PTEN is also the key mediator of the PI3K/Akt pathway in the neuronal response to zinc insult. We used primary cortical neurons and neuroblastoma N2a cells to show that zinc treatment results in a reduction of the PTEN protein level in parallel with increased NEDD4-1 gene/protein expression. The reduced PTEN level is associated with an activated PI3K pathway as determined by elevated phosphorylation of both Akt and GSK-3 as well as by the attenuating effect of a specific PI3K inhibitor (wortmannin). The reduction of PTEN can be attributed to increased protein degradation via the ubiquitin proteosomal system, as we show NEDD4-1 to be the major E3 ligase responsible for PTEN ubiquitination in neurons. Moreover, PTEN and NEDD4-1 appear to be able to counter-regulate each other to mediate the neuronal response to zinc. This reciprocal regulation requires the PI3K signaling pathway, suggesting a feedback loop mechanism. This study demonstrates that NEDD4-1-mediated PTEN ubiquitination is crucial in the regulation of PI3K/Akt signaling by PTEN during the neuronal response to zinc, which may represent a common mechanism in neurodegeneration.

Statin's excitoprotection is mediated by sAPP and the subsequent attenuation of calpain-induced truncation events, likely via rho-ROCK signaling.

The widely used cholesterol-lowering drugs, statins, were reported to reduce the incidence of stroke and the progression of Alzheimer's disease. However, little is known on how statins exert these beneficial effects. In this study, we investigated the molecular mechanisms underlying the neuroprotective actions of statins in primary cultured cortical neurons. We found that chronic treatment of neurons with a low dosage of two CNS-permeable statins (lovastatin and simvastatin) selectively reduced NMDA-induced cell death but not the caspase-mediated apoptosis. The protective effects of stains were inhibited by mevalonate, a PI3K inhibitor, and tyrphostin AG538, suggesting roles for cholesterol and insulin/IGF-1 signaling in the neurotoxic response. We further demonstrate that statins block calcium-dependent calpain activation, resulting in complete suppression of protein truncation events on multiple calpain substrates that are involved in neuronal death including CDK5 coactivator p35 cleavage to p25, GSK3 and beta-catenin. This is followed by reduced and increased nuclear translocation of p25 and beta-catenin, respectively. Under excitotoxic conditions, the activities of CDK5 and beta-catenin are exclusively regulated by calpain-mediated cleavage while apoptosis modulates beta-catenin mainly through phosphorylation. Strikingly, our data demonstrate that the calpain-blocking effect of statins is largely mediated by stimulation of alpha-secretase cleavage of APP, resulting in increased secretion of its soluble form, sAPP. Finally, our data suggest that statin-regulated sAPP secretion occurs via activation of the PI3K pathway and inhibition of ROCK signaling. Altogether, our study provides novel insights into statin-mediated neuronal excitoprotection through both cholesterol-dependent and -independent mechanisms and links them to calpain-mediated neuronal death.

Antidiabetic drug metformin (GlucophageR) increases biogenesis of Alzheimer's amyloid peptides via up-regulating BACE1 transcription.

Epidemiological, clinical and experimental evidence suggests a link between type 2 diabetes and Alzheimer's disease (AD). Insulin modulates metabolism of beta-amyloid precursor protein (APP) in neurons, decreasing the intracellular accumulation of beta-amyloid (Abeta) peptides, which are pivotal in AD pathogenesis. The present study investigates whether the widely prescribed insulin-sensitizing drug, metformin (Glucophage(R)), affects APP metabolism and Abeta generation in various cell models. We demonstrate that metformin, at doses that lead to activation of the AMP-activated protein kinase (AMPK), significantly increases the generation of both intracellular and extracellular Abeta species. Furthermore, the effect of metformin on Abeta generation is mediated by transcriptional up-regulation of beta-secretase (BACE1), which results in an elevated protein level and increased enzymatic activity. Unlike insulin, metformin exerts no effect on Abeta degradation. In addition, we found that glucose deprivation and various tyrphostins, known inhibitors of insulin-like growth factors/insulin receptor tyrosine kinases, do not modulate the effect of metformin on Abeta. Finally, inhibition of AMP-activated protein kinase (AMPK) by the pharmacological inhibitor Compound C largely suppresses metformin's effect on Abeta generation and BACE1 transcription, suggesting an AMPK-dependent mechanism. Although insulin and metformin display opposing effects on Abeta generation, in combined use, metformin enhances insulin's effect in reducing Abeta levels. Our findings suggest a potentially harmful consequence of this widely prescribed antidiabetic drug when used as a monotherapy in elderly diabetic patients.

Amyloid precursor protein is involved in staurosporine induced glial differentiation of neural progenitor cells.

Staurosporine (STS) has been reported as not only a pro-apoptotic agent, but also a terminal differentiation inducer in several neuroblastoma cell lines. Here, we report involvement of amyloid precursor protein (APP) in a STS induced astrocytic differentiation of human neural progenitor cells (NT-2/D1). We found that STS-treated NT-2/D1 cells expressed astrocyte-specific glial fibrillary acidic protein (GFAP), aspartate transporter, and glutamate transporter-1 with a distinctive astrocytic morphology. STS treatment increased GFAP promoter activity and increased expression and secretion of APP in NT-2/D1 cell culture. Overexpressed APP enhanced GFAP promoter activity and expression of GFAP, while gene silencing of APP by RNA interference decreased GFAP expression. These results indicate involvement of APP in STS induced astrocytic differentiation of NT-2/D1 cells. Furthermore, suppression of ERK1/2 phosphorylation, which is known to regulate APP expression by a MEK1 inhibitor, PD098059, reduced both APP and GFAP expression in STS treated NT-2/D1 cells. Thus, STS may induce astrocytic differentiation of NT-2/D1 by increasing APP levels associate with activation of ERK pathway.

Human zinc finger protein 161, a novel transcriptional activator of the dopamine transporter.

The dopamine transporter (DAT) terminates dopaminergic neurotransmission via reuptake of released dopamine into presynaptic neurons. We have cloned 2.5 kb of the regulatory region upstream of human DAT (hDAT) and constructed a series of deletion mutants to test promoter activity. A comparison of promoter activity between non-neural and neuronal cell lines reveals an interesting difference in pattern. In the PC12 cell line, activity of the proximal promoter is strongly silenced by one or more unidentified elements spanning positions -395 to -2465 of the hDAT gene. Our studies focus on identifying and characterizing the activating factor for hDAT transcription in the sequence between -2511 and -2492 (5(')-CTA CCT GCA CAG TTC ACG GA-3('), termed HY1). In this investigation, we cloned the zinc finger protein 161 (ZFP161) gene as a HY1-binding factor, using the yeast one-hybrid screen. Recombinant ZFP161 was produced to evaluate the DNA-binding properties of the protein. The ability of ZFP161 to directly bind HY1 was examined in an electrophoretic mobility shift assay. RT-PCR analyses revealed that transfection of ZFP161 induced hDAT mRNA expression in HEK293 cells. We additionally confirmed the expression and localization of the DAT protein, using a specific antibody. Both the HY1 sequence and the downstream region were necessary for activation of the hDAT promoter by ZFP161. This finding suggests that the site of cofactor interaction with ZFP161 may exist downstream of HY1.

RNA interference with small hairpin RNAs transcribed from a human U6 promoter-driven DNA vector.

RNA interference (RNAi), a process of sequence-specific gene suppression, has been known as a natural gene regulatory mechanism in a wide range of organisms. Recently, a small-interference RNA (siRNA) technology has been reported to produce post-transcriptional gene silencing in mammalian cells. In the present study, we constructed a human U6 promoter-driven mammalian expression vector to produce hairpin double-stranded RNA and transfected this into a human cell line. Using this siRNA system, we were able to knock down the gene expression of an enhanced green fluorescence protein. This result indicates that the plasmid vector-based siRNA system is a promising method to downregulate gene expression in human cells.