Thalidomide possesses two optical isomers which have been reported to exhibit different pharmacol... more Thalidomide possesses two optical isomers which have been reported to exhibit different pharmacological and toxicological activities. However, the precise mechanism by which the two isomers exert their different activities remains poorly understood. Here, we present structural and biochemical studies of (S)-and (R)-enantiomers bound to the primary target of thalidomide, cereblon (CRBN). Our biochemical studies employed deuterium-substituted thalidomides to suppress optical isomer conversion, and established that the (S)-enantiomer exhibited ~10-fold stronger binding to CRBN and inhibition of self-ubiquitylation compared to the (R)-enantiomer. The crystal structures of the thalidomide-binding domain of CRBN bound to each enantiomer show that both enantiomers bind the tri-Trp pocket, although the bound form of the (S)-enantiomer exhibited a more relaxed glutarimide ring conformation. The (S)-enantiomer induced greater teratogenic effects on fins of zebrafish compared to the (R)-enantiomer. This study has established a mechanism by which thalidomide exerts its effects in a stereospecific manner at the atomic level. More than 50 years have passed since thalidomide was first prescribed as a sedative and antiemetic to provide effective relief from morning sickness during early pregnancy. The teratogenic effects associated with its use were soon discovered along with severe birth defects such as phocomelia and amelia 1-3. Pharmacological studies aimed at delineating the cause of thalidomide-induced teratogenicity led to the discovery of a number of unexpected pharmacological activities including anti-inflammatory, inhibition of tumor necrosis factor (TNF)-α production and anti-angiogenic effects 4-6. Other studies demonstrated thalidomide-induced oxidative stress, which results in DNA damage or perturbation of the signaling pathways involving NF-κB or Bmp/Dkk1/Wnt 7-10. Thalidomide and its derivatives are now widely used as potent immunomodulatory drugs (IMiDs) in the treatment of several diseases including multiple myeloma (MM) and leprosy (Hansen's disease) 11-14. Furthermore, thalidomide has recently been investigated in the treatment of vascular diseases 15,16. Thalidomide is the small synthetic compound α-phthalimido-glutarimide (IUPAC systematic name, 3-(RS)-2-(2,6-dioxo-3-piperidyl)isoindole-1,3-dione), which possesses one chiral centre (the C3-carbon atom of the glutarimide ring), and comprises a racemic mixture of two optical isomers, (R)-(also (+)-) and (S)-(also (−)-) enantiomers, currently in clinical use. The (R)-and (S)-enantiomers were once thought to be responsible for the sedative and teratogenic effects, respectively. This idea was challenged by the findings that the (R)-enantiomer is also teratogenic in a rabbit model 11,12 and that interconversion of the enantiomers could occur under physiological conditions 17-19. However, a number of reports, including some describing the use of configuration-stable thalidomide analogues, have shown that the (S)-enantiomer is more teratogenic and effective at inhibiting TNF-α production and angiogenesis compared to the (R)-enantiomer 20-24. Thalidomide directly binds cereblon (CRBN), which was originally reported as a cerebral protein associated with mild mental retardation 25,26. CRBN is a highly conserved protein that forms a CRL4-type E3 ubiquitin ligase complex, CRL4 CRBN , with Cul4A and damaged DNA binding protein 1 (DDB1), and plays a key role in limb outgrowth and expression of fibroblast growth factor Fgf8 in zebrafish and chicks 25. Thalidomide is suggested to initiate its teratogenic effects by binding to CRBN and modulating the associated ubiquitin ligase activity 25. Moreover, a human MM cell line with deletion of the CRBN gene was shown to be resistant to thalidomide derivatives, indicating that CRBN is involved
Pomalidomide and lenalidomide are immunomodulatory agents that were derived from thalidomide. Cer... more Pomalidomide and lenalidomide are immunomodulatory agents that were derived from thalidomide. Cereblon (CRBN) is a common direct target of thalidomide and related compounds and works as a Cullin Ring 4 E3 ubiquitin ligase (CRL4) with DDB1, CUL4, and ROC1. The substrate specificity of CRL4 CRBN is modulated by thalidomide-related compounds. While lenalidomide is approved for the treatment of several diseases including multiple myeloma, 5q-syndrome, mantle cell lymphoma, and follicular lymphoma, pomalidomide is approved only for the treatment of lenalidomide-resistant multiple myeloma. Here we show that PLZF/ZBTB16 and its fusion proteins are pomalidomide-dependent neosubstrates of CRL4 CRBN. PLZF joins to RARα or potentially other partner genes, and the translocation causes leukemias, such as acute promyelocytic leukemia and T-cell acute lymphoblastic leukemia. We demonstrate that pomalidomide treatment induces PLZF-RARα degradation, resulting in antiproliferation of leukemic cells expressing PLZF-RARα. This study highlights a potential therapeutic role of pomalidomide as a degrader of leukemogenic fusion proteins.
The transcription factor ATF/E4TF3 stimulates transcription from the adenovirus early region 4 (E... more The transcription factor ATF/E4TF3 stimulates transcription from the adenovirus early region 4 (E4) promoter by binding to specific promoter elements. Among the multiple forms of ATF/E4TF3, two forms with molecular masses of 47 and 43 kDa, which are most active in transcription in vitro from the E4 promoter, have been purified to homogeneity from HeLa cells by sequence-specific DNA affinity chromatography and biochemically characterized. Each purified protein formed a homodimer. These two homodimers were easily altered into a heterodimer when mixed together in the absence, but not in the presence, of the specific DNA sequence. All of these dimers were able to activate transcription in vitro from the E4 promoter by binding to the specific DNA sequence. Their activities to bind to DNA or stimulate transcription were different. The ability of the 47-kDa homodimer to stimulate transcription in vitro from the E4 promoter was approximately nine and three times higher than the abilities of...
We examined the synthesis of early and late simian virus 40 (SV40) mRNA's in SV40-infected ce... more We examined the synthesis of early and late simian virus 40 (SV40) mRNA's in SV40-infected cells treated with two kinds of protein synthesis inhibitors. SV40 stimulated the synthesis of mRNA's for both large and small tumor antigens in cells pretreated with the drug emetine before the addition of virus. Emetine is a stringent inhibitor of protein synthesis and, thus, protein factors necessary for transcription and processing of these mRNA's probably preexist in the cell. Surprisingly, infection of cells pretreated with the protein synthesis inhibitor cycloheximide stimulated the synthesis of about 10-fold-higher levels of early viral mRNA's than did comparable infections of nontreated cells. This amplification of early viral mRNA steady-state levels is probably not due to inhibition of synthesis of the early A gene product since the same degree of drug-specific amplification was seen in SV40 tsA -infected cells that were cultured at the nonpermissive temperature. How...
The transcription factor E4TF1 stimulates transcription from the adenovirus early region 4 promot... more The transcription factor E4TF1 stimulates transcription from the adenovirus early region 4 promoter by binding to a specific promoter element. E4TF1 has been purified to homogeneity from HeLa cells by sequence-specific DNA affinity chromatography and characterized. E4TF1 is composed of at least two distinct subunits identified as 60 kd and 53 kd polypeptides. The 60 kd protein alone is able to bind to the specific DNA sequence but not to stimulate transcription in vitro. The 53 kd protein alone neither binds to DNA nor stimulates transcription in vitro. However, the 53 kd protein is able to interact with the 60 kd protein and the interaction confers the ability to stimulate transcription in vitro and to increase the DNA binding affinity of the 60 kd protein. This study provides evidence that the interaction between the two different subunits of E4TF1 is required for it to function as a transcription factor, and that one of the subunits binds to a specific DNA sequence and the other works as a modulator.
Thalidomide possesses two optical isomers which have been reported to exhibit different pharmacol... more Thalidomide possesses two optical isomers which have been reported to exhibit different pharmacological and toxicological activities. However, the precise mechanism by which the two isomers exert their different activities remains poorly understood. Here, we present structural and biochemical studies of (S)- and (R)-enantiomers bound to the primary target of thalidomide, cereblon (CRBN). Our biochemical studies employed deuterium-substituted thalidomides to suppress optical isomer conversion, and established that the (S)-enantiomer exhibited ~10-fold stronger binding to CRBN and inhibition of self-ubiquitylation compared to the (R)-enantiomer. The crystal structures of the thalidomide-binding domain of CRBN bound to each enantiomer show that both enantiomers bind the tri-Trp pocket, although the bound form of the (S)-enantiomer exhibited a more relaxed glutarimide ring conformation. The (S)-enantiomer induced greater teratogenic effects on fins of zebrafish compared to the (R)-enant...
A series of deletion mutants spanning the adenovirus early region IV (EIV) promoter were tested f... more A series of deletion mutants spanning the adenovirus early region IV (EIV) promoter were tested for transcription activity in vitro. At least three elements were found to be important for maximal transcription in HeLa whole-cell extracts. Deletion of the TATA box drastically reduced the transcription activity from the EIV promoter. Sequences between nucleotides -58 and -44 are also important for efficient transcription since deletion of this region reduced activity by 50%. More importantly, sequences residing upstream from -140 critically influence the level of EIV transcription. Deletion of sequences between nucleotides -325 (the right terminus of adenovirus genome) and -140 reduced the level of transcription more than 10-fold. It is possible that a specific cellular factor stimulates EIV transcription by recognition of these upstream sequences. The dependence of transcription from the EIV promoter on a distal upstream element may explain some aspects of the regulation of this prom...
Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et... more Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980) were tested for their efficiency and fidelity in directing protein synthesis in reticulocyte lysates. Simian virus 40 deoxyribonucleic acid (DNA), cleaved by various restriction endonucleases, was used as the template. Successful translation of the small tumor antigen t, as well as the capsid proteins VP1, VP2, and VP3, was detected by immunoprecipitation analysis. Although no synthesis of large T antigen was detected, use of this technology allows detection of large T synthesis resulting from the correct splicing of as little as 0.2% of the in vitro RNA transcripts, making it ideal for use as an in vitro splicing assay. Transcripts synthesized in vitro were used as messages at least as efficiently as were viral messenger RNA's (mRNA's) synthesized in vivo; and in the case of small t, there was more efficient translation of ...
Wefound that the expression vector previously constructed using the promoter of the GAL7 gene of ... more Wefound that the expression vector previously constructed using the promoter of the GAL7 gene of Saccharomyces cerevisiae lacked the transcription terminator. The addition of the GAL7 terminator resulted in a more than 3-fold increase in the production of the protein encoded by CDNAof early region la (Ela) of human adenovirus, without affecting the amount of its mRNA. The positive regulatory gene, GAL4,when amplified with a vector ofa high copy number, caused an about 5-fold increase in Ela production. Wealso found that medium containing a mixture of galactose and glucose can be used for the efficient production of the Ela protein.
6-((2-(Dimethylamino)ethyl)amino)-3-hydroxy-7H-indeno(2,1-c)-quinolin-7-one dihydrochloride (TAS-... more 6-((2-(Dimethylamino)ethyl)amino)-3-hydroxy-7H-indeno(2,1-c)-quinolin-7-one dihydrochloride (TAS-103) is a quinoline derivative that displays antitumor activity in murine and human tumor models. TAS-103 has been reported to be a potent topoisomerase II poison. However, other studies have indicated that cellular susceptibility to TAS-103 is not correlated with topoisomerase II expression. Because the direct target of TAS-103 remained unclear, we searched for a TAS-103 binding protein using high-performance affinity latex beads. We obtained a component of the signal recognition particle (SRP) as a TAS-103 binding protein. This component is a 54-kDa subunit (SRP54) of SRP, which mediates the proper delivery of secretory proteins in cells. We fractioned 293T cell lysates using gel-filtration chromatography and performed a coimmunoprecipitation assay using 293T cells expressing FLAG-tagged SRP54. The results revealed that TAS-103 disrupts SRP complex formation and reduces the amount of S...
Mediator complexes are required for activators to stimulate Pol II preinitiation complex assembly... more Mediator complexes are required for activators to stimulate Pol II preinitiation complex assembly on an associated promoter. We show here that for the mouse Egr1 gene, controlled largely by MAP kinase phosphorylation of the ELK1 transcription factor, the MED23 Mediator subunit that interacts with phospho-ELK1 is also required to stimulate Pol II initiation at a step subsequent to preinitiation complex assembly. In Med23-/- cells, histone acetylation, methylation, and chromatin remodeling complex association at the Egr1 promoter were equivalent to that of wild-type cells, yet Egr1 induction was greatly reduced. MAP kinase activation stimulated Pol II and GTF promoter binding. However, the difference in factor binding between wild-type and mutant cells was much less than the difference in transcription, and Pol II remained localized to the promoter in mutant cells. These results indicate that an interaction with MED23 stimulates initiation by promoter bound Pol II in addition to Pol I...
Thalidomide Teratogenicity Target In the late 1950s and early 1960s, thalidomide was prescribed t... more Thalidomide Teratogenicity Target In the late 1950s and early 1960s, thalidomide was prescribed to pregnant women as a cure for morning sickness, but it was then found to have developmental defects, most obviously, stunted limbs in thousands of babies. Although its use was banned worldwide, thalidomide has since been found to be a valuable treatment for a range of cancers, inflammatory disorders, and leprosy. Several hypotheses have been proposed, but the mechanism of action of thalidomide is unknown. Using zebrafish and chicken as animal models, Ito et al. (p. 1345 ) show that the protein cereblon is a primary target of thalidomide. Thalidomide exerts teratogenic effects by binding to cereblon and inhibiting associated enzymatic activity important for limb development. Knowing the mechanism of action of thalidomide should encourage the search for thalidomide derivatives without teratogenic activity.
In the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides the synthesis of compone... more In the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides the synthesis of components of the photosystem is regulated in response to oxygen tension and light intensity. We have purified and cloned a transacting protein (SPB) that binds to the promoter region of the pu/operon, which encodes the apoproteins of light-harvesting complex I and the reaction center. The SPB was composed of a single polypeptide with an apparent molecular mass of 15.0 kDa. The nucleotide sequence of the spb gene was determined. The gene encoded 104 amino acid residues, which correspond to a molecular mass of 11.5 kDa. SPB exhibited 53% homology to HvrA in Rhodobacter capsulatus. The deduced amino acid sequence indicated that SPB contained a region with homology to the leucinezipper motif of c-JUN, a transcription factor in eukaryotes, and SPB also had a DNA-binding domain on the amino-terminal side of the leucine-zipper motif. The leucine-zipper motif of SPB might contribute to the formation of a dimer. Northern analysis indicated that spb was constitutively and monocistronically transcribed in R. sphaeroides, irrespective of growth conditions. Structural and functional differences between SPB and HvrA are discussed.
Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridiz... more Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3 0-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.
The TBP-like protein (TLP/TRF2/TLF), which belongs to the TBP family of proteins, is present in a... more The TBP-like protein (TLP/TRF2/TLF), which belongs to the TBP family of proteins, is present in all metazoan organisms. Although the human TLP has been reported to interfere with transcription from TATA-containing promoters, the transcription activation potential of TLP in higher animals is obscure. We previously demonstrated that arti®cially promoter-recruited TLP behaves like an unconventional transcriptional activator. In this study, we investigated the effects of TLP on TATAless promoters of mouse and human terminal deoxynucleotidyl transferase (TdT) genes by transient reporter assays. As expected, TLP repressed both basal and activator-augmented transcription from the TATA-containing adenovirus major late promoter (MLP) and E1B promoter. On the other hand, however, TLP signi®cantly stimulated both basal and activated transcription from TdT promoters. We investigated the strength of the promoters in chicken DT40 cells that lack the TLP gene. The MLP showed higher activity but the TdT promoter showed lower activity in TLP-null cells than in the wild-type cells. Moreover, ectopic expression of mouse TLP in the TLP-null cells considerably stimulated the TdT promoter. Insertion of a TATA element upstream from the TdT core promoter resulted in a loss of TLP-mediated activation. The mouse TLP was demonstrated to bind speci®cally to TFIIA with greater strength than TBP. We constructed mutated TLPs having amino acid substitutions that impair TFIIA binding. A representative TLP mutant lacking TFIIA-binding ability could not stimulate transcription from the TdT promoter, whereas that mutation suppressed TLP-mediated transcription repression of TATA promoters. The results of the present study suggest that the vertebrate TLP potentiates exogenous TATA-less promoters and that TFIIA plays an important role in the TLP function.
NELF and DSIF act together to inhibit transcription elongation in vitro, and are implicated in ca... more NELF and DSIF act together to inhibit transcription elongation in vitro, and are implicated in causing promoter proximal pausing on the hsp70 gene in Drosophila. Here, further characterization of Drosophila NELF is provided. Drosophila NELF has four subunits similar to subunits of human NELF. The amino acid sequences of NELF-B and NELF-D are highly conserved throughout their lengths, while NELF-A and NELF-E contain nonconserved regions inserted between conserved N-and Cterminal regions. Immunodepletion of NELF or DSIF from a nuclear extract desensitizes transcription in vitro to DRB. Immunodepletion of NELF also impairs promoter proximal pausing on the hsp70 promoter in vitro without affecting initiation. Chromatin immunoprecipitation analyses detect NELF at the promoters of the hsp70 and b1-tubulin genes where promoter proximal pausing has been previously detected. Heat shock induction of hsp70 results in a marked decrease in NELF at the hsp70 promoter. Immunofluorescence analysis of polytene chromosomes shows extensive colocalization of the NELF-B and NELF-D subunits at hundreds of interbands. Neither subunit appears to be recruited to puffs. These results provide a foundation for genetic and biochemical analysis of NELF in Drosophila.
Physiological and pharmacological processes mediated by glucocorticoids involve tissue- and conte... more Physiological and pharmacological processes mediated by glucocorticoids involve tissue- and context-specific regulation of glucocorticoid-responsive gene expression via glucocorticoid receptor (GR). However, the molecular mechanisms underlying such highly coordinated regulation of glucocorticoid actions remain to be studied. We here addressed this issue using atp1a1 and scnn1a, both of which are up-regulated in response to corticosteroids in human embryonic kidney-derived 293 cells, but resistant in liver-derived HepG2 cells. Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) represses gene expression via, at least, two distinct mechanisms, i.e. positive transcription elongation factor b sequestration and direct interaction with GR, and is relatively high in HepG2 cells compared with 293 cells. Given this, we focused on the role of HEXIM1 in transcriptional regulation of these GR target genes. In HepG2 cells, hormone resistance of atp1a1 and scnn1a was diminished by either knoc...
Negative elongation factor (NELF) is a four subunit transcription elongation factor that has been... more Negative elongation factor (NELF) is a four subunit transcription elongation factor that has been implicated in numerous diseases ranging from neurological disorders to cancer. Here we show that NELF interacts with the nuclear cap binding complex (CBC), a multifunctional factor that plays important roles in several mRNA processing steps, and the two factors together participate in the 3 0 end processing of replication-dependent histone mRNAs, most likely through association with the histone stem-loop binding protein (SLBP). Strikingly, absence of NELF and CBC causes aberrant production of polyadenylated histone mRNAs. Moreover, NELF is physically associated with histone gene loci and forms distinct intranuclear foci that we call NELF bodies, which often overlap with Cajal bodies and cleavage bodies. Our results point to a surprising role of NELF in the 3 0 end processing of histone mRNAs and also suggest that NELF is a new factor that coordinates different mRNA processing steps during transcription.
Regulatory mechanisms controlling the timing of developmental events are crucial for proper devel... more Regulatory mechanisms controlling the timing of developmental events are crucial for proper development to occur. ftz-f1 is expressed in a temporally regulated manner following pulses of ecdysteroid and this precise expression is necessary for the development of Drosophila melanogaster . To understand how insect hormone ecdysteroids regulate the timing of FTZ-F1 expression, we purified a DNA binding regulator of ftz-f1 . Mass spectroscopy analysis revealed this protein to be a fly homolog of mammalian B lymphocyte-induced maturation protein 1 (Blimp-1). Drosophila Blimp-1 ( dBlimp-1 ) is induced directly by 20-hydroxyecdysone, and its product exists during high-ecdysteroid periods and turns over rapidly. Forced expression of dBlimp-1 and RNA interference analysis indicate that dBlimp-1 acts as a repressor and controls the timing of FTZ-F1 expression. Furthermore, its prolonged expression results in delay of pupation timing. These results suggest that the transient transcriptional re...
AF5q31 (also called MCEF ) was identified by its involvement in chromosomal translocation with th... more AF5q31 (also called MCEF ) was identified by its involvement in chromosomal translocation with the gene MLL (mixed lineage leukemia), which is associated with infant acute lymphoblastic leukemia. Several potential roles have been proposed for AF5q31 and other family genes, but the specific requirements of AF5q31 during development remain unclear. Here, we show that AF5q31 is essential for spermatogenesis. Although most AF5q31-deficient mice died in utero and neonatally with impaired embryonic development and shrunken alveoli, respectively, 13% of AF5q31-deficient mice thrived as wild-type mice did. However, the male mice were sterile with azoospermia. Histological examinations revealed the arrest of germ cell development at the stage of spermiogenesis, and virtually no spermatozoa were seen in the epididymis. AF5q31 was found to be preferentially expressed in Sertoli cells. Furthermore, mutant mice displayed severely impaired expression of protamine 1, protamine 2, and transition pr...
Thalidomide possesses two optical isomers which have been reported to exhibit different pharmacol... more Thalidomide possesses two optical isomers which have been reported to exhibit different pharmacological and toxicological activities. However, the precise mechanism by which the two isomers exert their different activities remains poorly understood. Here, we present structural and biochemical studies of (S)-and (R)-enantiomers bound to the primary target of thalidomide, cereblon (CRBN). Our biochemical studies employed deuterium-substituted thalidomides to suppress optical isomer conversion, and established that the (S)-enantiomer exhibited ~10-fold stronger binding to CRBN and inhibition of self-ubiquitylation compared to the (R)-enantiomer. The crystal structures of the thalidomide-binding domain of CRBN bound to each enantiomer show that both enantiomers bind the tri-Trp pocket, although the bound form of the (S)-enantiomer exhibited a more relaxed glutarimide ring conformation. The (S)-enantiomer induced greater teratogenic effects on fins of zebrafish compared to the (R)-enantiomer. This study has established a mechanism by which thalidomide exerts its effects in a stereospecific manner at the atomic level. More than 50 years have passed since thalidomide was first prescribed as a sedative and antiemetic to provide effective relief from morning sickness during early pregnancy. The teratogenic effects associated with its use were soon discovered along with severe birth defects such as phocomelia and amelia 1-3. Pharmacological studies aimed at delineating the cause of thalidomide-induced teratogenicity led to the discovery of a number of unexpected pharmacological activities including anti-inflammatory, inhibition of tumor necrosis factor (TNF)-α production and anti-angiogenic effects 4-6. Other studies demonstrated thalidomide-induced oxidative stress, which results in DNA damage or perturbation of the signaling pathways involving NF-κB or Bmp/Dkk1/Wnt 7-10. Thalidomide and its derivatives are now widely used as potent immunomodulatory drugs (IMiDs) in the treatment of several diseases including multiple myeloma (MM) and leprosy (Hansen's disease) 11-14. Furthermore, thalidomide has recently been investigated in the treatment of vascular diseases 15,16. Thalidomide is the small synthetic compound α-phthalimido-glutarimide (IUPAC systematic name, 3-(RS)-2-(2,6-dioxo-3-piperidyl)isoindole-1,3-dione), which possesses one chiral centre (the C3-carbon atom of the glutarimide ring), and comprises a racemic mixture of two optical isomers, (R)-(also (+)-) and (S)-(also (−)-) enantiomers, currently in clinical use. The (R)-and (S)-enantiomers were once thought to be responsible for the sedative and teratogenic effects, respectively. This idea was challenged by the findings that the (R)-enantiomer is also teratogenic in a rabbit model 11,12 and that interconversion of the enantiomers could occur under physiological conditions 17-19. However, a number of reports, including some describing the use of configuration-stable thalidomide analogues, have shown that the (S)-enantiomer is more teratogenic and effective at inhibiting TNF-α production and angiogenesis compared to the (R)-enantiomer 20-24. Thalidomide directly binds cereblon (CRBN), which was originally reported as a cerebral protein associated with mild mental retardation 25,26. CRBN is a highly conserved protein that forms a CRL4-type E3 ubiquitin ligase complex, CRL4 CRBN , with Cul4A and damaged DNA binding protein 1 (DDB1), and plays a key role in limb outgrowth and expression of fibroblast growth factor Fgf8 in zebrafish and chicks 25. Thalidomide is suggested to initiate its teratogenic effects by binding to CRBN and modulating the associated ubiquitin ligase activity 25. Moreover, a human MM cell line with deletion of the CRBN gene was shown to be resistant to thalidomide derivatives, indicating that CRBN is involved
Pomalidomide and lenalidomide are immunomodulatory agents that were derived from thalidomide. Cer... more Pomalidomide and lenalidomide are immunomodulatory agents that were derived from thalidomide. Cereblon (CRBN) is a common direct target of thalidomide and related compounds and works as a Cullin Ring 4 E3 ubiquitin ligase (CRL4) with DDB1, CUL4, and ROC1. The substrate specificity of CRL4 CRBN is modulated by thalidomide-related compounds. While lenalidomide is approved for the treatment of several diseases including multiple myeloma, 5q-syndrome, mantle cell lymphoma, and follicular lymphoma, pomalidomide is approved only for the treatment of lenalidomide-resistant multiple myeloma. Here we show that PLZF/ZBTB16 and its fusion proteins are pomalidomide-dependent neosubstrates of CRL4 CRBN. PLZF joins to RARα or potentially other partner genes, and the translocation causes leukemias, such as acute promyelocytic leukemia and T-cell acute lymphoblastic leukemia. We demonstrate that pomalidomide treatment induces PLZF-RARα degradation, resulting in antiproliferation of leukemic cells expressing PLZF-RARα. This study highlights a potential therapeutic role of pomalidomide as a degrader of leukemogenic fusion proteins.
The transcription factor ATF/E4TF3 stimulates transcription from the adenovirus early region 4 (E... more The transcription factor ATF/E4TF3 stimulates transcription from the adenovirus early region 4 (E4) promoter by binding to specific promoter elements. Among the multiple forms of ATF/E4TF3, two forms with molecular masses of 47 and 43 kDa, which are most active in transcription in vitro from the E4 promoter, have been purified to homogeneity from HeLa cells by sequence-specific DNA affinity chromatography and biochemically characterized. Each purified protein formed a homodimer. These two homodimers were easily altered into a heterodimer when mixed together in the absence, but not in the presence, of the specific DNA sequence. All of these dimers were able to activate transcription in vitro from the E4 promoter by binding to the specific DNA sequence. Their activities to bind to DNA or stimulate transcription were different. The ability of the 47-kDa homodimer to stimulate transcription in vitro from the E4 promoter was approximately nine and three times higher than the abilities of...
We examined the synthesis of early and late simian virus 40 (SV40) mRNA's in SV40-infected ce... more We examined the synthesis of early and late simian virus 40 (SV40) mRNA's in SV40-infected cells treated with two kinds of protein synthesis inhibitors. SV40 stimulated the synthesis of mRNA's for both large and small tumor antigens in cells pretreated with the drug emetine before the addition of virus. Emetine is a stringent inhibitor of protein synthesis and, thus, protein factors necessary for transcription and processing of these mRNA's probably preexist in the cell. Surprisingly, infection of cells pretreated with the protein synthesis inhibitor cycloheximide stimulated the synthesis of about 10-fold-higher levels of early viral mRNA's than did comparable infections of nontreated cells. This amplification of early viral mRNA steady-state levels is probably not due to inhibition of synthesis of the early A gene product since the same degree of drug-specific amplification was seen in SV40 tsA -infected cells that were cultured at the nonpermissive temperature. How...
The transcription factor E4TF1 stimulates transcription from the adenovirus early region 4 promot... more The transcription factor E4TF1 stimulates transcription from the adenovirus early region 4 promoter by binding to a specific promoter element. E4TF1 has been purified to homogeneity from HeLa cells by sequence-specific DNA affinity chromatography and characterized. E4TF1 is composed of at least two distinct subunits identified as 60 kd and 53 kd polypeptides. The 60 kd protein alone is able to bind to the specific DNA sequence but not to stimulate transcription in vitro. The 53 kd protein alone neither binds to DNA nor stimulates transcription in vitro. However, the 53 kd protein is able to interact with the 60 kd protein and the interaction confers the ability to stimulate transcription in vitro and to increase the DNA binding affinity of the 60 kd protein. This study provides evidence that the interaction between the two different subunits of E4TF1 is required for it to function as a transcription factor, and that one of the subunits binds to a specific DNA sequence and the other works as a modulator.
Thalidomide possesses two optical isomers which have been reported to exhibit different pharmacol... more Thalidomide possesses two optical isomers which have been reported to exhibit different pharmacological and toxicological activities. However, the precise mechanism by which the two isomers exert their different activities remains poorly understood. Here, we present structural and biochemical studies of (S)- and (R)-enantiomers bound to the primary target of thalidomide, cereblon (CRBN). Our biochemical studies employed deuterium-substituted thalidomides to suppress optical isomer conversion, and established that the (S)-enantiomer exhibited ~10-fold stronger binding to CRBN and inhibition of self-ubiquitylation compared to the (R)-enantiomer. The crystal structures of the thalidomide-binding domain of CRBN bound to each enantiomer show that both enantiomers bind the tri-Trp pocket, although the bound form of the (S)-enantiomer exhibited a more relaxed glutarimide ring conformation. The (S)-enantiomer induced greater teratogenic effects on fins of zebrafish compared to the (R)-enant...
A series of deletion mutants spanning the adenovirus early region IV (EIV) promoter were tested f... more A series of deletion mutants spanning the adenovirus early region IV (EIV) promoter were tested for transcription activity in vitro. At least three elements were found to be important for maximal transcription in HeLa whole-cell extracts. Deletion of the TATA box drastically reduced the transcription activity from the EIV promoter. Sequences between nucleotides -58 and -44 are also important for efficient transcription since deletion of this region reduced activity by 50%. More importantly, sequences residing upstream from -140 critically influence the level of EIV transcription. Deletion of sequences between nucleotides -325 (the right terminus of adenovirus genome) and -140 reduced the level of transcription more than 10-fold. It is possible that a specific cellular factor stimulates EIV transcription by recognition of these upstream sequences. The dependence of transcription from the EIV promoter on a distal upstream element may explain some aspects of the regulation of this prom...
Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et... more Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980) were tested for their efficiency and fidelity in directing protein synthesis in reticulocyte lysates. Simian virus 40 deoxyribonucleic acid (DNA), cleaved by various restriction endonucleases, was used as the template. Successful translation of the small tumor antigen t, as well as the capsid proteins VP1, VP2, and VP3, was detected by immunoprecipitation analysis. Although no synthesis of large T antigen was detected, use of this technology allows detection of large T synthesis resulting from the correct splicing of as little as 0.2% of the in vitro RNA transcripts, making it ideal for use as an in vitro splicing assay. Transcripts synthesized in vitro were used as messages at least as efficiently as were viral messenger RNA's (mRNA's) synthesized in vivo; and in the case of small t, there was more efficient translation of ...
Wefound that the expression vector previously constructed using the promoter of the GAL7 gene of ... more Wefound that the expression vector previously constructed using the promoter of the GAL7 gene of Saccharomyces cerevisiae lacked the transcription terminator. The addition of the GAL7 terminator resulted in a more than 3-fold increase in the production of the protein encoded by CDNAof early region la (Ela) of human adenovirus, without affecting the amount of its mRNA. The positive regulatory gene, GAL4,when amplified with a vector ofa high copy number, caused an about 5-fold increase in Ela production. Wealso found that medium containing a mixture of galactose and glucose can be used for the efficient production of the Ela protein.
6-((2-(Dimethylamino)ethyl)amino)-3-hydroxy-7H-indeno(2,1-c)-quinolin-7-one dihydrochloride (TAS-... more 6-((2-(Dimethylamino)ethyl)amino)-3-hydroxy-7H-indeno(2,1-c)-quinolin-7-one dihydrochloride (TAS-103) is a quinoline derivative that displays antitumor activity in murine and human tumor models. TAS-103 has been reported to be a potent topoisomerase II poison. However, other studies have indicated that cellular susceptibility to TAS-103 is not correlated with topoisomerase II expression. Because the direct target of TAS-103 remained unclear, we searched for a TAS-103 binding protein using high-performance affinity latex beads. We obtained a component of the signal recognition particle (SRP) as a TAS-103 binding protein. This component is a 54-kDa subunit (SRP54) of SRP, which mediates the proper delivery of secretory proteins in cells. We fractioned 293T cell lysates using gel-filtration chromatography and performed a coimmunoprecipitation assay using 293T cells expressing FLAG-tagged SRP54. The results revealed that TAS-103 disrupts SRP complex formation and reduces the amount of S...
Mediator complexes are required for activators to stimulate Pol II preinitiation complex assembly... more Mediator complexes are required for activators to stimulate Pol II preinitiation complex assembly on an associated promoter. We show here that for the mouse Egr1 gene, controlled largely by MAP kinase phosphorylation of the ELK1 transcription factor, the MED23 Mediator subunit that interacts with phospho-ELK1 is also required to stimulate Pol II initiation at a step subsequent to preinitiation complex assembly. In Med23-/- cells, histone acetylation, methylation, and chromatin remodeling complex association at the Egr1 promoter were equivalent to that of wild-type cells, yet Egr1 induction was greatly reduced. MAP kinase activation stimulated Pol II and GTF promoter binding. However, the difference in factor binding between wild-type and mutant cells was much less than the difference in transcription, and Pol II remained localized to the promoter in mutant cells. These results indicate that an interaction with MED23 stimulates initiation by promoter bound Pol II in addition to Pol I...
Thalidomide Teratogenicity Target In the late 1950s and early 1960s, thalidomide was prescribed t... more Thalidomide Teratogenicity Target In the late 1950s and early 1960s, thalidomide was prescribed to pregnant women as a cure for morning sickness, but it was then found to have developmental defects, most obviously, stunted limbs in thousands of babies. Although its use was banned worldwide, thalidomide has since been found to be a valuable treatment for a range of cancers, inflammatory disorders, and leprosy. Several hypotheses have been proposed, but the mechanism of action of thalidomide is unknown. Using zebrafish and chicken as animal models, Ito et al. (p. 1345 ) show that the protein cereblon is a primary target of thalidomide. Thalidomide exerts teratogenic effects by binding to cereblon and inhibiting associated enzymatic activity important for limb development. Knowing the mechanism of action of thalidomide should encourage the search for thalidomide derivatives without teratogenic activity.
In the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides the synthesis of compone... more In the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides the synthesis of components of the photosystem is regulated in response to oxygen tension and light intensity. We have purified and cloned a transacting protein (SPB) that binds to the promoter region of the pu/operon, which encodes the apoproteins of light-harvesting complex I and the reaction center. The SPB was composed of a single polypeptide with an apparent molecular mass of 15.0 kDa. The nucleotide sequence of the spb gene was determined. The gene encoded 104 amino acid residues, which correspond to a molecular mass of 11.5 kDa. SPB exhibited 53% homology to HvrA in Rhodobacter capsulatus. The deduced amino acid sequence indicated that SPB contained a region with homology to the leucinezipper motif of c-JUN, a transcription factor in eukaryotes, and SPB also had a DNA-binding domain on the amino-terminal side of the leucine-zipper motif. The leucine-zipper motif of SPB might contribute to the formation of a dimer. Northern analysis indicated that spb was constitutively and monocistronically transcribed in R. sphaeroides, irrespective of growth conditions. Structural and functional differences between SPB and HvrA are discussed.
Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridiz... more Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3 0-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.
The TBP-like protein (TLP/TRF2/TLF), which belongs to the TBP family of proteins, is present in a... more The TBP-like protein (TLP/TRF2/TLF), which belongs to the TBP family of proteins, is present in all metazoan organisms. Although the human TLP has been reported to interfere with transcription from TATA-containing promoters, the transcription activation potential of TLP in higher animals is obscure. We previously demonstrated that arti®cially promoter-recruited TLP behaves like an unconventional transcriptional activator. In this study, we investigated the effects of TLP on TATAless promoters of mouse and human terminal deoxynucleotidyl transferase (TdT) genes by transient reporter assays. As expected, TLP repressed both basal and activator-augmented transcription from the TATA-containing adenovirus major late promoter (MLP) and E1B promoter. On the other hand, however, TLP signi®cantly stimulated both basal and activated transcription from TdT promoters. We investigated the strength of the promoters in chicken DT40 cells that lack the TLP gene. The MLP showed higher activity but the TdT promoter showed lower activity in TLP-null cells than in the wild-type cells. Moreover, ectopic expression of mouse TLP in the TLP-null cells considerably stimulated the TdT promoter. Insertion of a TATA element upstream from the TdT core promoter resulted in a loss of TLP-mediated activation. The mouse TLP was demonstrated to bind speci®cally to TFIIA with greater strength than TBP. We constructed mutated TLPs having amino acid substitutions that impair TFIIA binding. A representative TLP mutant lacking TFIIA-binding ability could not stimulate transcription from the TdT promoter, whereas that mutation suppressed TLP-mediated transcription repression of TATA promoters. The results of the present study suggest that the vertebrate TLP potentiates exogenous TATA-less promoters and that TFIIA plays an important role in the TLP function.
NELF and DSIF act together to inhibit transcription elongation in vitro, and are implicated in ca... more NELF and DSIF act together to inhibit transcription elongation in vitro, and are implicated in causing promoter proximal pausing on the hsp70 gene in Drosophila. Here, further characterization of Drosophila NELF is provided. Drosophila NELF has four subunits similar to subunits of human NELF. The amino acid sequences of NELF-B and NELF-D are highly conserved throughout their lengths, while NELF-A and NELF-E contain nonconserved regions inserted between conserved N-and Cterminal regions. Immunodepletion of NELF or DSIF from a nuclear extract desensitizes transcription in vitro to DRB. Immunodepletion of NELF also impairs promoter proximal pausing on the hsp70 promoter in vitro without affecting initiation. Chromatin immunoprecipitation analyses detect NELF at the promoters of the hsp70 and b1-tubulin genes where promoter proximal pausing has been previously detected. Heat shock induction of hsp70 results in a marked decrease in NELF at the hsp70 promoter. Immunofluorescence analysis of polytene chromosomes shows extensive colocalization of the NELF-B and NELF-D subunits at hundreds of interbands. Neither subunit appears to be recruited to puffs. These results provide a foundation for genetic and biochemical analysis of NELF in Drosophila.
Physiological and pharmacological processes mediated by glucocorticoids involve tissue- and conte... more Physiological and pharmacological processes mediated by glucocorticoids involve tissue- and context-specific regulation of glucocorticoid-responsive gene expression via glucocorticoid receptor (GR). However, the molecular mechanisms underlying such highly coordinated regulation of glucocorticoid actions remain to be studied. We here addressed this issue using atp1a1 and scnn1a, both of which are up-regulated in response to corticosteroids in human embryonic kidney-derived 293 cells, but resistant in liver-derived HepG2 cells. Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) represses gene expression via, at least, two distinct mechanisms, i.e. positive transcription elongation factor b sequestration and direct interaction with GR, and is relatively high in HepG2 cells compared with 293 cells. Given this, we focused on the role of HEXIM1 in transcriptional regulation of these GR target genes. In HepG2 cells, hormone resistance of atp1a1 and scnn1a was diminished by either knoc...
Negative elongation factor (NELF) is a four subunit transcription elongation factor that has been... more Negative elongation factor (NELF) is a four subunit transcription elongation factor that has been implicated in numerous diseases ranging from neurological disorders to cancer. Here we show that NELF interacts with the nuclear cap binding complex (CBC), a multifunctional factor that plays important roles in several mRNA processing steps, and the two factors together participate in the 3 0 end processing of replication-dependent histone mRNAs, most likely through association with the histone stem-loop binding protein (SLBP). Strikingly, absence of NELF and CBC causes aberrant production of polyadenylated histone mRNAs. Moreover, NELF is physically associated with histone gene loci and forms distinct intranuclear foci that we call NELF bodies, which often overlap with Cajal bodies and cleavage bodies. Our results point to a surprising role of NELF in the 3 0 end processing of histone mRNAs and also suggest that NELF is a new factor that coordinates different mRNA processing steps during transcription.
Regulatory mechanisms controlling the timing of developmental events are crucial for proper devel... more Regulatory mechanisms controlling the timing of developmental events are crucial for proper development to occur. ftz-f1 is expressed in a temporally regulated manner following pulses of ecdysteroid and this precise expression is necessary for the development of Drosophila melanogaster . To understand how insect hormone ecdysteroids regulate the timing of FTZ-F1 expression, we purified a DNA binding regulator of ftz-f1 . Mass spectroscopy analysis revealed this protein to be a fly homolog of mammalian B lymphocyte-induced maturation protein 1 (Blimp-1). Drosophila Blimp-1 ( dBlimp-1 ) is induced directly by 20-hydroxyecdysone, and its product exists during high-ecdysteroid periods and turns over rapidly. Forced expression of dBlimp-1 and RNA interference analysis indicate that dBlimp-1 acts as a repressor and controls the timing of FTZ-F1 expression. Furthermore, its prolonged expression results in delay of pupation timing. These results suggest that the transient transcriptional re...
AF5q31 (also called MCEF ) was identified by its involvement in chromosomal translocation with th... more AF5q31 (also called MCEF ) was identified by its involvement in chromosomal translocation with the gene MLL (mixed lineage leukemia), which is associated with infant acute lymphoblastic leukemia. Several potential roles have been proposed for AF5q31 and other family genes, but the specific requirements of AF5q31 during development remain unclear. Here, we show that AF5q31 is essential for spermatogenesis. Although most AF5q31-deficient mice died in utero and neonatally with impaired embryonic development and shrunken alveoli, respectively, 13% of AF5q31-deficient mice thrived as wild-type mice did. However, the male mice were sterile with azoospermia. Histological examinations revealed the arrest of germ cell development at the stage of spermiogenesis, and virtually no spermatozoa were seen in the epididymis. AF5q31 was found to be preferentially expressed in Sertoli cells. Furthermore, mutant mice displayed severely impaired expression of protamine 1, protamine 2, and transition pr...
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Papers by Hiroshi Handa