Involvement of homeobox transcription factor Mohawk in palatogenesis.

Palatogenesis is affected by many factors, including gene polymorphisms and exposure to toxic chemicals during sensitive developmental periods. Cleft palate is one of the most common congenital anomalies, and ongoing efforts to elucidate the molecular mechanisms underlying palatogenesis are providing useful insights to reduce the risk of this disorder. To identify novel potential regulators of palatogenesis, we analyzed public transcriptome datasets from a mouse model of cleft palate caused by selective deletion of transforming growth factor-ß (TGFß) receptor type 2 in cranial neural crest cells. We identified the homeobox transcription factor Mohawk (Mkx) as a gene downregulated in the maxilla of TGFß knockout mice compared with wild-type mice. To examine the role of mkx in palatogenesis, we used CRISPR/Cas9 editing to generate zebrafish with impaired expression of mkxa and mkxb, the zebrafish homologs of Mkx. We found that mkx crispants expressed reduced levels of gli1, a critical transcription factor in the Sonic hedgehog (SHH) signaling pathway that plays an important role in the regulation of palatogenesis. Furthermore, we found that mkxa-/- zebrafish were more susceptible than mkxa+/+ zebrafish to the deleterious effects of cyclopamine, an inhibitor of SHH signaling, on upper jaw development. These results suggest that Mkx may be involved in palatogenesis regulated by TGFß and SHH signaling, and that impairment in Mkx function may be related to the etiology of cleft palate.

Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis.

Hepatic apoptosis is involved in a variety of pathophysiologic conditions in the liver, including hepatitis, steatosis, and drug-induced liver injury. The development of easy-to-perform and reliable in vivo assays would thus greatly enhance the efforts to understand liver diseases and identify associated genes and potential drugs. In this study, we developed a transgenic zebrafish line that was suitable for the assessment of caspase 3 activity in the liver by using in vivo fluorescence imaging. The larvae of transgenic zebrafish dominantly expressed Casper3GR in the liver under control of the promoter of the phosphoenolpyruvate carboxykinase 1 gene. Casper3GR is composed of two fluorescent proteins, tagGFP and tagRFP, which are connected via a peptide linker that can be cleaved by activated caspase 3. Under tagGFP excitation conditions in zebrafish that were exposed to the well-characterized hepatotoxicant isoniazid, we detected increased and decreased fluorescence associated with tagGFP and tagRFP, respectively. This result suggests that isoniazid activates caspase 3 in the zebrafish liver, which digests the linker between tagGFP and tagRFP, resulting in a reduction in the Förster resonance energy transfer to tagRFP upon tagGFP excitation. We also detected isoniazid-induced inhibition of caspase 3 activity in zebrafish that were treated with the hepatoprotectants ursodeoxycholic acid and obeticholic acid. The transgenic zebrafish that were developed in this study could be a powerful tool for identifying both hepatotoxic and hepatoprotective drugs, as well as for analyzing the effects of the genes of interest to hepatic apoptosis.

Genetic Engineering of Carbon Monoxide-dependent Hydrogen-producing Machinery in Parageobacillus thermoglucosidasius.

The metabolic engineering of carbon monoxide (CO) oxidizers has the potential to create efficient biocatalysts to produce hydrogen and other valuable chemicals. We herein applied markerless gene deletion to CO dehydrogenase/energy-converting hydrogenase (CODH/ECH) in the thermophilic facultative anaerobe, Parageobacillus thermoglucosidasius. We initially compared the transformation efficiency of two strains, NBRC 107763T and TG4. We then disrupted CODH, ECH, and both enzymes in NBRC 107763T. The characterization of growth in all three disruptants under 100% CO demonstrated that both enzymes were essential for CO-dependent growth with hydrogen production in P. thermoglucosidasius. The present results will become a platform for the further metabolic engineering of this organism.

Effects of audio and visual distraction on patients' vital signs and tolerance during esophagogastroduodenoscopy: a randomized controlled trial.

BACKGROUND: Esophagogastroduodenoscopy (EGD) provides an indispensable and unambiguous inspection allowing the discovery upper gastrointestinal lesions. However, many patients are anxious about undergoing EGD. Few studies have investigated the influence on patients' vital signs and tolerance during EGD using subjective and objective assessments. This study was a prospective randomized controlled study that investigated the influence of audio and visual distraction on EGD. METHODS: We randomly divided 289 subjects who underwent EGD into 4 groups (control group, audio group, visual group, combination group) and examined their vital signs, heart rate variability (HRV), psychological items, and acceptance of distraction. RESULTS: Pulse rate (PR) at post-distraction and post-EGD in the 3 distraction groups were significantly lower than those of control group (p <  0.001 and p <  0.01, respectively). Blood pressure (BP) during and post-EGD was significantly higher than that at pre-EGD in control group (p <  0.05), but no significant elevation of BP was observed during the latter half of EGD and post-EGD in the 3 distraction groups. BP at post-distraction improved significantly compared to pre-distraction in the 3 distraction groups (p <  0.05). There was a significant difference in the low-frequency (LF) power/ high-frequency (HF) power at post-distraction and post-EGD among the 4 groups (p <  0.001 and p <  0.001, respectively). The LF power/HF power at post-distraction and post-EGD in the 3 distraction groups was significantly lower than that in control group (p <  0.05). Several items of profile of mood states (POMS) and the impression of EGD at post-distraction improved significantly compared to those at pre-distraction among the 3 distraction groups (p <  0.05). Visual analog scale (VAS) of willingness for the next use of distraction in the 3 distraction groups was excellent because VAS was more than 70. CONCLUSIONS: Distractions effectively improved psychological factors, vital signs and some of HRV at pre and post-EGD. Distractions may suppress BP elevation during the latter half of EGD and lead to stability of HRV on EGD. TRIAL REGISTRATION: This prospective trial was registered in the University Hospital Medical Information Network (UMIN) Clinical Trials Registry as UMIN000029637. Registered on 20 October 2017.

C3orf70 Is Involved in Neural and Neurobehavioral Development.

Neurogenesis is the process by which undifferentiated progenitor cells develop into mature and functional neurons. Defects in neurogenesis are associated with neurodevelopmental and neuropsychiatric disorders; therefore, elucidating the molecular mechanisms underlying neurogenesis can advance our understanding of the pathophysiology of these disorders and facilitate the discovery of novel therapeutic targets. In this study, we performed a comparative transcriptomic analysis to identify common targets of the proneural transcription factors Neurog1/2 and Ascl1 during neurogenesis of human and mouse stem cells. We successfully identified C3orf70 as a novel common target gene of Neurog1/2 and Ascl1 during neurogenesis. Using in situ hybridization, we demonstrated that c3orf70a and c3orf70b, two orthologs of C3orf70, were expressed in the midbrain and hindbrain of zebrafish larvae. We generated c3orf70 knockout zebrafish using CRISPR/Cas9 technology and demonstrated that loss of c3orf70 resulted in significantly decreased expression of the mature neuron markers elavl3 and eno2. We also found that expression of irx3b, a zebrafish ortholog of IRX3 and a midbrain/hindbrain marker, was significantly reduced in c3orf70 knockout zebrafish. Finally, we demonstrated that neurobehaviors related to circadian rhythm and altered light-dark conditions were significantly impaired in c3orf70 knockout zebrafish. These results suggest that C3orf70 is involved in neural and neurobehavioral development and that defects in C3orf70 may be associated with midbrain/hindbrain-related neurodevelopmental and neuropsychiatric disorders.

Generation of a Triple-Transgenic Zebrafish Line for Assessment of Developmental Neurotoxicity during Neuronal Differentiation.

: The developing brain is extremely sensitive to many chemicals. Exposure to neurotoxicants during development has been implicated in various neuropsychiatric and neurological disorders, including autism spectrum disorders and schizophrenia. Various screening methods have been used to assess the developmental neurotoxicity (DNT) of chemicals, with most assays focusing on cell viability, apoptosis, proliferation, migration, neuronal differentiation, and neuronal network formation. However, assessment of toxicity during progenitor cell differentiation into neurons, astrocytes, and oligodendrocytes often requires immunohistochemistry, which is a reliable but labor-intensive and time-consuming assay. Here, we report the development of a triple-transgenic zebrafish line that expresses distinct fluorescent proteins in neurons (Cerulean), astrocytes (mCherry), and oligodendrocytes (mCitrine), which can be used to detect DNT during neuronal differentiation. Using in vivo fluorescence microscopy, we could detect DNT by 6 of the 10 neurotoxicants tested after exposure to zebrafish from 12 h to 5 days' post-fertilization. Moreover, the chemicals could be clustered into three main DNT groups based on the fluorescence pattern: (i) inhibition of neuron and oligodendrocyte differentiation and stimulation of astrocyte differentiation; (ii) inhibition of neuron and oligodendrocyte differentiation; and (iii) inhibition of neuron and astrocyte differentiation, which suggests that reporter expression reflects the toxicodynamics of the chemicals. Thus, the triple-transgenic zebrafish line developed here may be a useful tool to assess DNT during neuronal differentiation.

The influence of various distractions prior to upper gastrointestinal endoscopy: a prospective randomized controlled study.

BACKGROUND: Although many patients still have anxiety about upper gastrointestinal (GI) endoscopy, there have been few reports on the influence of distractions for a person who is going to undergo upper GI endoscopy soon. This study was a prospective randomized controlled study investigating the influence of distractions, such as auditive and visual distractions using subjective and objective assessments including autonomic nervous function prior to upper GI endoscopy. METHODS: 206 subjects who underwent upper GI endoscopy as regular health check-ups were divided randomly into 4 groups prior to upper GI endoscopy; group 1 (control group), group 2 (auditive group), group 3 (visual group), and group 4 (combination group). We measured vital signs, autonomic nervous function, profile of mood state (POMS), and the impression for upper GI endoscopy pre- and post-distraction in the 4 groups. RESULTS: There was no significant difference in vital signs between 5 and 15 min after sitting in group 1, however, several vital signs in all distraction groups improved significantly after distraction (Pulse rate (P): p <  0.001 in group 4; blood pressure: p <  0.05 in group 2, 3, 4) and the rate of decrease in P and diastolic blood pressure was highest in group 4 (p <  0.001). Several scores of POMS and the impression for upper GI endoscopy post-distraction improved significantly compared to pre-distraction between distraction groups and the satisfaction for distraction was highest in group 4 (p <  0.01). Regarding autonomic nerve function, the low- frequency power/ high- frequency power ratio post-distraction was significantly lower than that pre-distraction in all distraction groups (p <  0.001). CONCLUSIONS: Although auditive distraction alone and visual distraction alone were effective, a combination distraction was more effective than any other distraction by subjective and objective assessments. These distractions, which were simple and safe, may play an assistive role in the stability of physical and psychological conditions prior to upper GI endoscopy. TRIAL REGISTRATION: This trial was registered in the University Hospital Medical Information Network (UMIN) Clinical Trials Registry as UMIN000022801 . Registered on 10 July 2016.

The novel heme-dependent inducible protein, SRRD regulates heme biosynthesis and circadian rhythms.

Heme plays a role in the regulation of the expression of genes related to circadian rhythms and heme metabolism. In order to identify new heme-regulated proteins, an RNA sequence analysis using mouse NIH3T3 cells treated without or with 5-aminolevulinic acid (ALA) was performed. Among the changes observed in the levels of various mRNAs including heme oxygenase-1 (HO-1) and ALA synthase-1 (ALAS1), a mouse homologue of the plant circadian-regulating protein SRR1, SRR1 domain containing (SRRD) was induced by the ALA treatment. The expression of SRRD was dependent on heme biosynthesis, and increased the production of heme. SRRD was expressed under circadian rhythms, and influenced the expression of clock genes including PER2, BMAL1, and CLOCK. The knockout of SRRD arrested the growth of cells, indicating that SRRD plays roles in heme-regulated circadian rhythms and cell proliferation.

Potential role of heme metabolism in the inducible expression of heme oxygenase-1.

BACKGROUND: The degradation of heme significantly contributes to cytoprotective effects against oxidative stress and inflammation. The enzyme heme oxygenase-1 (HO-1), involved in the degradation of heme, forms carbon monoxide (CO), ferrous iron, and bilirubin in conjunction with biliverdin reductase, and is induced by various stimuli including oxidative stress and heavy metals. We examined the involvement of heme metabolism in the induction of HO-1 by the inducers sulforaphane and sodium arsenite. METHODS: We examined the expression of HO-1 in sulforaphane-, sodium arsenite- and CORM3-treated HEK293T cells, by measuring the transcriptional activity and levels of mRNA and protein. RESULTS: The blockade of heme biosynthesis by succinylacetone and N-methyl protoporphyrin, which are inhibitors of heme biosynthesis, markedly decreased the induction of HO-1. The knockdown of the first enzyme in the biosynthesis of heme, 5-aminolevulinic acid synthase, also decreased the induction of HO-1. The cessation of HO-1 induction occurred at the transcriptional and translational levels, and was mediated by the activation of the heme-binding transcriptional repressor Bach1 and translational factor HRI. CO appeared to improve the expression of HO-1 at the transcriptional and translational levels. CONCLUSIONS: We demonstrated the importance of heme metabolism in the stress-inducible expression of HO-1, and also that heme and its degradation products are protective factors for self-defense responses. GENERAL SIGNIFICANCE: The key role of heme metabolism in the stress-inducible expression of HO-1 may promote further studies on heme and its degradation products as protective factors of cellular stresses and iron homeostasis in specialized cells, organs, and whole animal systems.

Calreticulin discriminates the proximal region at the N-glycosylation site of Glc1Man9GlcNAc2 ligand.

Calreticulin (CRT) is well known as a lectin-like chaperone that recognizes Glc1Man9GlcNAc2 (G1M9)-glycoproteins in the endoplasmic reticulum (ER). However, whether CRT can directly interact with the aglycone moiety (protein portion) of the glycoprotein remains controversial. To improve our understanding of CRT interactions, structure-defined G1M9-derivatives with different aglycones (-OH, -Gly-NH2, and -Gly-Glu-(t)Bu) were used as CRT ligands, and their interactions with recombinant CRT were analyzed using thermal shift analysis. The results showed that CRT binds strongly to a G1M9-ligand in the order -Gly-Glu-(t)Bu > -Gly-NH2 > -OH, which is the same as that of the reglucosylation of Man9GlcNAc2 (M9)-derivatives by the folding sensor enzyme UGGT (UDP-glucose: glycoprotein glucosyltransferase). Our results indicate that, similar to UGGT, CRT discriminates the proximal region at the N-glycosylation site, suggesting a similar mechanism mediating the recognition of aglycone moieties in the ER glycoprotein quality control system.

p53 directly regulates the transcription of the human frataxin gene and its lack of regulation in tumor cells decreases the utilization of mitochondrial iron.

Mitochondrial frataxin functions in iron homeostasis, biogenesis of iron-sulfur clusters, protection from oxidative stress and apoptosis, and as a tumor suppressor protein. We examined regulation of the expression of the human frataxin by p53. Pifithrin-α, an inhibitor of p53 function, and knockdown of p53 decreased the level of frataxin mRNA in human kidney HEK 293T cells. The transcriptional activity of the human frataxin gene is enhanced by the proximal promoter containing the p53-responsive element (p53RE) on the gene. Chromatin immunoprecipitation assay and electrophoretic mobility shift assay confirmed the binding of p53 to the human frataxin p53RE. The expression of wild-type p53 in human cancer HeLa cells increased the reporter activity carrying p53RE at the region of -209 to -200bp of the frataxin promoter. Finally, when the HeLa cells overexpressing frataxin were treated with 5-aminolevulinic acid (ALA), there was less accumulation of protoporphyrin than HeLa control cells, and it was sharply decreased by the addition of iron citrate, suggesting that the utilization of mitochondrial iron for heme biosynthesis can be dependent on the level of frataxin. Alternatively, the low expression of frataxin not regulated by p53 in tumor cells lowers the utilization of iron in mitochondria, causing the tumor-specific ALA-induced accumulation of protoporphyrin.

Long-term adaptation of the Bombyx mori BmN4 cell line to grow in serum-free culture.

Bombyx mori ovary-derived BmN4 cells have been successfully adapted to a commercial serum-free medium (SFM; SF900-II) by gradually reducing the serum-containing TC-100 medium content from 100 to 0% (v/v). The BmN4 cells adapted to the SFM (BmN-SFM) adhered strongly to the culture flask and showed altered cell morphology. The BmN-SFM was subcultured 200 times, and the population doubling time was 4.70 d. Infection studies showed that BmN-SFM cells were easily susceptible to B. mori nucleopolyhedrovirus (BmNPV), and both the multiplication of budded virus and the promoter activity of the polyhedrin gene in BmN-SFM cells were almost the same as those in BmN4 cells before adaptation. Additionally, mouse interleukin-3 expressed by a recombinant BmNPV was normally secreted and modified with N-linked glycans in BmN-SFM cells. These findings indicate that BmN-SFM is particularly useful for a BmNPV-based baculovirus expression vector system with serum-free conditions.

Neurotransmitter transporter family including SLC6A6 and SLC6A13 contributes to the 5-aminolevulinic acid (ALA)-induced accumulation of protoporphyrin IX and photodamage, through uptake of ALA by cancerous cells.

δ-Aminolevulinic acid (ALA)-induced protoporphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy (PDT). To clarify the mechanisms of ALA uptake by tumor cells, we have examined the ALA-induced accumulation of protoporphyrin by the treatment of colon cancer DLD-1 and epithelial cancer HeLa cells with γ-aminobutyric acid (GABA)-related compounds. When the cells were treated with GABA, taurine and ß-alanine, the level of protoporphyrin was decreased, suggesting that plasma membrane transporters involved in the transport of neurotransmitters contribute to the uptake of ALA. By transfection with neurotransmitter transporters SLC6A6, SLC6A8 and SLC6A13 cDNA, the ALA- and ALA methylester-dependent accumulation of protoporphyrin markedly increased in HEK293T cells, dependent on an increase in the uptake of ALA. When ALA-treated cells were exposed to white light, the extent of photodamage increased in SLC6A6- and SLC6A13-expressing cells. Conversely, knockdown of SLC6A6 or SLC6A13 with siRNAs in DLD-1 and HeLa cells decreased the ALA-induced accumulation. The expression of SLC6A6 and SLC6A13 was found in some cancer cell lines. Immunohistochemical studies revealed that the presence of these transporters was elevated in colon cancerous cells. These results indicated that neurotransmitter transporters including SLC6A6 and SLC6A13 mediate the uptake of ALA and can play roles in the enhancement of ALA-induced accumulation of protoporphyrin in cancerous cells.

The enzyme engineering of mutant homodimer and heterodimer of coproporphyinogen oxidase contributes to new insight into hereditary coproporphyria and harderoporphyria.

Hereditary coproporphyria (HCP) is an autosomal dominant-inherited disease of haem biosynthesis caused by partial deficiency of the enzyme coproporphyrinogen oxidase (CPOX). Patients with HCP show <50% of normal activity and those with the rare autosomal recessive harderoporphyria accumulate harderoporphyrinogen, an intermediate porphyrin of the CPOX reaction. To clarify the relationship of the low enzyme activity with these diseases, we expressed mutant CPOX carrying His-tag from these porphyria patients and co-expressed mutant CPOX carrying His-tag and normal CPOX carrying HA-tag in a tandem fashion in Escherichia coli. Purification of the His-tag-containing enzyme revealed that the His-enzyme forms a heterodimer in association with the HA-enzyme, and analysis using a cross-link reagent confirmed that the enzyme is a dimer (∼70 kDa). Then, we expressed homo- and heterodimers composed of the wild-type (wt) and engineered mutants of the enzyme or mutants from HCP patients. The monomer form of mutated CPOX did not show any activity and homodimeric enzymes derived from HCP mutant showed low activity (<20% of the control). Some mutations of amino acids 401-404 were associated with marked accumulation of harderoporphyrinogen, with a decrease in the production of protoporphyrinogen, whereas K404E derived from patients with harderoporphyria produced less harderoporphyrinogen. The heterodimers with wt and mutated subunits from HCP patients showed low protoporphyrinogen producing activity. These results show that the substitution of amino acids from R401 to K404 results in extremely low enzyme activity with either mutant homodimer or heterodimers containing normal and mutated subunits and can be linked to HCP disease.

The SNARE protein SYP71 expressed in vascular tissues is involved in symbiotic nitrogen fixation in Lotus japonicus nodules.

Soluble N-Ethylmaleimide Sensitive Factor Attachment Protein Receptor (SNARE) proteins are crucial for signal transduction and development in plants. Here, we investigate a Lotus japonicus symbiotic mutant defective in one of the SNARE proteins. When in symbiosis with rhizobia, the growth of the mutant was retarded compared with that of the wild-type plant. Although the mutant formed nodules, these exhibited lower nitrogen fixation activity than the wild type. The rhizobia were able to invade nodule cells, but enlarged symbiosomes were observed in the infected cells. The causal gene, designated LjSYP71 (for L. japonicus syntaxin of plants71), was identified by map-based cloning and shown to encode a Qc-SNARE protein homologous to Arabidopsis (Arabidopsis thaliana) SYP71. LjSYP71 was expressed ubiquitously in shoot, roots, and nodules, and transcripts were detected in the vascular tissues. In the mutant, no other visible defects in plant morphology were observed. Furthermore, in the presence of combined nitrogen, the mutant plant grew almost as well as the wild type. These results suggest that the vascular tissues expressing LjSYP71 play a pivotal role in symbiotic nitrogen fixation in L. japonicus nodules.

Properties of Jack bean α-mannosidase in the presence of hyaluronan.

An enzymatic reaction within a mesh-like structure constructed using hyaluronan was investigated in order to understand the influence of specific reaction environments in a living body on the reaction. This mesh-like structure, which mimicked extracellular matrix conditions, was found to accelerate glycohydrolysis by Jack bean α-mannosidase.

Translation of partially overlapping psbD-psbC mRNAs in chloroplasts: the role of 5'-processing and translational coupling.

The chloroplast psbD and psbC genes encode the D2 and CP43 proteins of the photosystem II complex, and they are generally cotranscribed. We report studies on the basic translation process of tobacco psbD-psbC mRNAs using an in vitro translation system from tobacco chloroplasts. The primary transcript has an unusually long 5'-UTR (905 nt). We show that it is translatable. Processing of the 5'-UTR greatly enhances the translation efficiency of the psbD cistron. A striking feature is that psbD and psbC cistrons overlap by 14 nt. Removal of the psbD 5'-UTR plus the start codon and introduction of a premature termination codon in the psbD cistron considerably reduce the translation efficiency of the downstream psbC cistron. These results indicate that translation of the psbC cistron depends largely on that of the upstream psbD cistron and thus shows translational coupling; however, a portion is independently translated. These observations, together with the presence of monocistronic psbC mRNAs, suggest that the psbD and psbC cistrons are translated via multiple processes to produce necessary amounts of D2 and CP43 proteins.

Chronic actinic dermatitis in a patient with adult T-cell leukemia.

A patient with chronic actinic dermatitis showed a leukocytosis with peripheral atypical lymphocytes. Histology of cutaneous eruptions disclosed a dense infiltrate of lymphocytes mixed with large hyperchromatic cells in the dermis and Pautrier-like microabscesses in the epidermis. However, clonal rearrangements of the T-cell receptors were not detected. On the other hand, monoclonal integration of human T-cell lymphotrophic virus type I provirus was found in the peripheral lymphocytes. In the present case, pseudolymphomatous changes occurred in association with adult T-cell leukemia. Southern blot analyses were useful for the correct diagnosis.

Clinical effect of vitamin D3 analogues is not inactivated by subsequent UV exposure.

BACKGROUND: Combining phototherapy with topical vitamin D3 analogues is a useful therapy for the treatment of psoriasis by reducing the cumulative UV dose required for clearance of lesions. Experimental investigations demonstrated that calcipotriol is degraded by UV radiation, and suggested that calcipotriol should be applied after phototherapy but not immediately before. METHODS: Calcipotriol or maxacalcitol ointment was topically applied to psoriatic plaques of six patients immediately before or after phototherapy on the right or left side of the body, respectively. RESULTS: Topical application of vitamin D3 analogues either before or after irradiation by psoralen and UVA radiation (PUVA) or narrow-band (NB)-UVB showed exactly similar effects in all patients. CONCLUSION: Therapeutic effects of vitamin D3 analogues are not clinically inactivated by subsequent irradiation with PUVA or NB-UVB phototherapy.

hDREF regulates cell proliferation and expression of ribosomal protein genes.

Although ribosomal proteins (RPs) are essential cellular constituents in all living organisms, mechanisms underlying regulation of their gene expression in mammals remain unclear. We have established that 22 out of 79 human RP genes contain sequences similar to the human DREF (DNA replication-related element-binding factor; hDREF) binding sequence (hDRE) within 200-bp regions upstream of their transcriptional start sites. Electrophoretic gel mobility shift assays and chromatin immunoprecipitation analysis indicated that hDREF binds to hDRE-like sequences in the RP genes both in vitro and in vivo. In addition, transient luciferase assays revealed that hDRE-like sequences act as positive elements for RP gene transcription and cotransfection of an hDREF-expressing plasmid was found to stimulate RP gene promoter activity. Like that of hDREF, expression of RP genes is increased during the late G(1) to S phases, and depletion of hDREF using short hairpin RNA-mediated knockdown decreased RP gene expression and cell proliferation in normal human fibroblasts. Knockdown of the RPS6 gene also resulted in impairment of cell proliferation. These data suggest that hDREF is an important transcription factor for cell proliferation which plays roles in cell cycle-dependent regulation of a number of RP genes.