N. Tanese - Academia.edu (original) (raw)

Papers by N. Tanese

Scientific reports, 2011

Transport of mRNAs to diverse neuronal locations via RNA granules serves an important function in... more Transport of mRNAs to diverse neuronal locations via RNA granules serves an important function in regulating protein synthesis within restricted sub-cellular domains. We recently detected the Huntington's disease protein huntingtin (Htt) in dendritic RNA granules; however, the functional significance of this localization is not known. Here we report that Htt and the huntingtin-associated protein 1 (HAP1) are co-localized with the microtubule motor proteins, the KIF5A kinesin and dynein, during dendritic transport of β-actin mRNA. Live cell imaging demonstrated that β-actin mRNA is associated with Htt, HAP1, and dynein intermediate chain in cultured neurons. Reduction in the levels of Htt, HAP1, KIF5A, and dynein heavy chain by lentiviral-based shRNAs resulted in a reduction in the transport of β-actin mRNA. These findings support a role for Htt in participating in the mRNA transport machinery that also contains HAP1, KIF5A, and dynein.

Cytometry Part A, 2012

The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase TrkB s... more The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase TrkB serve important regulatory roles for multiple aspects of the biology of neurons including cell death, survival, growth, differentiation, and plasticity. Regulation of the local availability of BDNF/TrkB at distinct subcellular domains such as soma, dendrites, axons, growth cones, nerve terminals, and spines appears to contribute to their specific functions. In view of the variance in size and shape of neurons and their compartments, previous quantitative studies of the BDNF/ TrkB protein and mRNA lacked a robust normalization procedure. To overcome this problem, we have established methods that use immunofluorescence detection of α-tubulin as a normalization factor for the quantitative analysis of protein and mRNA in primary rat cortical and striatal neurons in culture. The efficacy of this approach is demonstrated by studying the dynamic distribution of proteins and mRNA at different growth stages or conditions. Treatment of cultured neurons with KCl resulted in increased levels of TrkB protein, reduced levels of BDNF mRNA (composite of multiple transcripts) and a slight reduction in BDNF protein levels in the dendrites from the cortex. The KCl treatment also lowered the percentage of BDNF and TrkB proteins in the soma indicative of protein transport. Finally, analysis of the rat cortical and striatal neurons demonstrated comparable or even higher levels of BDNF/TrkB protein and BDNF mRNA in the neurons from the striatum. Thus, in contrast to previous observations made in vivo, striatal neurons are capable of synthesizing BDNF mRNA when cultured in growth media in vitro. The analytical approach presented here provides a detailed understanding of BDNF/TrkB levels in response to a variety of neuronal activities. Our methods could be used broadly, including applications in cell and tissue cytometry, to yield accurate quantitative data of gene expression in cellular and subcellular contexts.

Journal of Huntington's disease, Jan 29, 2016

The Huntington's disease (HD) protein huntingtin (Htt) plays a role in multiple cellular path... more The Huntington's disease (HD) protein huntingtin (Htt) plays a role in multiple cellular pathways. Deregulation of one or more of these pathways by the mutant Htt protein has been suggested to contribute to the disease pathogenesis. Our recent discovery-based proteomics studies have uncovered RNA binding proteins and translation factors associated with the endogenous Htt protein purified from mouse brains, suggesting a potential new role for Htt in RNA transport and translation. To investigate how Htt might affect RNA metabolism we set out to purify and analyze RNA associated with Htt. RNA was extracted from immunopurified Htt-containing protein complexes and analyzed by microarrays and RNA-Seq. Surprisingly, the most enriched mRNA that co-purified with Htt was Htt mRNA itself. The association of Htt protein and Htt mRNA was detected independent of intact ribosomes suggesting that it is not an RNA undergoing translation. Furthermore, we identified the recently reported mis-splic...

Proceedings of the National Academy of Sciences, 2008

Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansio... more Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansion of polyglutamines in the huntingtin (Htt) protein, whose cellular function remains controversial. To gain insight into Htt function, we purified epitope-tagged Htt and identified Argonaute as associated proteins. Colocalization studies demonstrated Htt and Ago2 to be present in P bodies, and depletion of Htt showed compromised RNA-mediated gene silencing. Mouse striatal cells expressing mutant Htt showed fewer P bodies and reduced reporter gene silencing activity compared with wild-type counterparts. These data suggest that the previously reported transcriptional deregulation in HD may be attributed in part to mutant Htt's role in post-transcriptional processes.

Molecular and Cellular Biology, 2010

The mammalian SWI/SNF chromatin-remodeling complex facilitates DNA access by transcription factor... more The mammalian SWI/SNF chromatin-remodeling complex facilitates DNA access by transcription factors and the transcription machinery. The characteristic member of human SWI/SNF-A is BAF250/ARID1, of which there are two isoforms, BAF250a/ARID1a and BAF250b/ARID1b. Here we report that BAF250b complexes purified from mammalian cells contain elongin C (Elo C), a BC box binding component of an E3 ubiquitin ligase. BAF250b was found to have a BC box motif, associate with Elo C in a BC box-dependent manner, and, together with cullin 2 and Roc1, assemble into an E3 ubiquitin ligase. The BAF250b BC box mutant protein was unstable in vivo and was autoubiquitinated in a manner similar to that for the VHL BC box mutants. The discovery that BAF250 is part of an E3 ubiquitin ligase adds an enzymatic function to the chromatin-remodeling complex SWI/SNF-A. The immunopurified BAF250b E3 ubiquitin ligase was found to target histone H2B at lysine 120 for monoubiquitination in vitro . To date, all H2B mo...

EMBO Journal, Dec 15, 1993

Regulation of transcription initiation by RNA polymerase II requires TFIID, a multisubunit comple... more Regulation of transcription initiation by RNA polymerase II requires TFIID, a multisubunit complex composed of the TATA binding protein (TBP) and at least seven tightly associated factors (TAFs). Some TAFs act as direct targets or coactivators for promoter-specific activators while others serve as interfaces for TAF-TAF interactions. Here, we report the molecular cloning, expression and characterization of Drosophila dTAFII60 and its human homolog, hTAFII70. Recombinant TAFII60/70 binds weakly to TBP and tightly to the largest subunit of TFIID, TAFII250. In the presence of TAFII60/70, TBP and TAFII250, a stable ternary complex is formed. Both the human and Drosophila proteins directly interact with another TFIID subunit, dTAFII40. Our findings reveal that Drosophila TAFII60 and human TAFII70 share a high degree of structural similarity and that their interactions with other subunits of TFIID are conserved.

Virology, 1989

An insertion mutation in the pal gene of Moloney murine leukemia virus (M-MuLV) was found to rend... more An insertion mutation in the pal gene of Moloney murine leukemia virus (M-MuLV) was found to render the virus temperature-sensitive for replication. A provirus containing a 12-bp insertion at the boundary between the reverse transcriptase (RT) and integrase (IN) domains induced the formation of mutant virions containing a partially processed RT-IN fusion protein. Some proteolytic processing to form mature RT and IN was observed at 32", but only aberrantly processed proteins were detected at 39". The uncleaved precursor was found to exhibit DNA polymerase activity, even though it could not support replication of the virus in viva at 39".

The Journal of biological chemistry, Jan 5, 1985

Expression of a region of the Moloney murine leukemia virus (M-MuLV) pol gene in Escherichia coli... more Expression of a region of the Moloney murine leukemia virus (M-MuLV) pol gene in Escherichia coli resulted in the synthesis of reverse transcriptase activity which could be detected in crude extracts. Construction of deletions at the 3' terminus of this gene resulted in a 4-fold increase in the level of the reverse transcriptase activity in the soluble fraction of crude lysates and yielded the high level production of a stable protein species of Mr = 71,000. Purification of this protein by column chromatography on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, and hydroxylapatite indicated that it was a multifunctional enzyme containing RNase H and reverse transcriptase activity. The Mr = 71,000 species had a sedimentation coefficient of 4.65 S by glycerol gradient centrifugation, indicating that the enzyme was a monomer. Using poly(A)+ mRNAs primed with oligo(dT), the enzyme synthesized double-stranded DNA copies between 1.3 and 9.9 kilobases in length. Synth...

Briefings in functional genomics, 2010

While initiation of transcription has attracted the most attention in the field of gene regulatio... more While initiation of transcription has attracted the most attention in the field of gene regulation, it has become clear that additional stages in the gene expression cascade including post-transcriptional events are under equally exquisite control. The seminal discovery that short RNAs (microRNA, small interfering RNA, Piwi-interacting RNA), play important roles in repressing gene expression has spurred a rush of new interest in post-transcriptional gene silencing mechanisms. The development of affinity tags and high-resolution tandem mass spectrometry (MS/MS) has greatly simplified the analysis of proteins that regulate gene expression. Further, the use of DNA microarrays and 'second generation' nucleic acid sequencing ('deep sequencing') technologies has facilitated the identification of their regulatory targets. These technological advancements mark a significant step towards a comprehensive understanding of gene regulatory networks. The purpose of this review is ...

Nature genetics, 2000

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding p... more At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration.

Molecular and cellular biology, 1998

TFIID is a multiprotein complex consisting of the TATA box binding protein and multiple tightly a... more TFIID is a multiprotein complex consisting of the TATA box binding protein and multiple tightly associated proteins (TAFIIs) that are required for transcription by selected activators. We previously reported cloning and partial characterization of human TAFII130 (hTAFII130). The central domain of hTAFII130 contains four glutamine-rich regions, designated Q1 to Q4, that are involved in interactions with the transcriptional activator Sp1. Mutational analysis has revealed specific regions within the glutamine-rich (Q1 to Q4) central region of hTAFII130 that are required for interaction with distinct activation domains. We tested amino- and carboxyl-terminal deletions of hTAFII130 for interaction with Sp1 activation domains A and B (Sp1A and Sp1B) and the N-terminal activation domain of CREB (CREB-N) by using the yeast two-hybrid system. Our results indicate that Sp1B interacts almost exclusively with the Q1 region of hTAFII130. In contrast, Sp1A makes multiple contacts with Q1 to Q4 of...

Journal of virology, 1997

The human cytomegalovirus (HCMV) major immediate-early (IE) proteins IEP86 (IE2(579aa)) and IEP72... more The human cytomegalovirus (HCMV) major immediate-early (IE) proteins IEP86 (IE2(579aa)) and IEP72 (IE1(491aa)) can transcriptionally activate a variety of simple promoters containing a TATA element and one upstream transcription factor binding site. In our previous studies, transcriptional activation was shown to correlate with IEP86 binding to both the TATA-box binding protein (TBP) and the transcription factor bound upstream. IEP72 often synergistically affects the activation by IEP86, although it has not previously been shown to directly interact in vitro with IEP86, TBP, or transcription factors (e.g., Sp1 and Tef-1) bound by IEP86. We report biochemical and genetic evidence suggesting that the major IE proteins may perform a function similar to that of the TBP-associated factors (TAFs) which make up TFIID. Consistent with this model, we found that the major IE proteins interact with a number of TAFs. In vitro, IEP86 bound with drosophila TAF(II)110 (dTAF(II)110) and human TAF(I...

The EMBO journal, Jan 15, 1993

Regulation of transcription initiation by RNA polymerase II requires TFIID, a multisubunit comple... more Regulation of transcription initiation by RNA polymerase II requires TFIID, a multisubunit complex composed of the TATA binding protein (TBP) and at least seven tightly associated factors (TAFs). Some TAFs act as direct targets or coactivators for promoter-specific activators while others serve as interfaces for TAF-TAF interactions. Here, we report the molecular cloning, expression and characterization of Drosophila dTAFII60 and its human homolog, hTAFII70. Recombinant TAFII60/70 binds weakly to TBP and tightly to the largest subunit of TFIID, TAFII250. In the presence of TAFII60/70, TBP and TAFII250, a stable ternary complex is formed. Both the human and Drosophila proteins directly interact with another TFIID subunit, dTAFII40. Our findings reveal that Drosophila TAFII60 and human TAFII70 share a high degree of structural similarity and that their interactions with other subunits of TFIID are conserved.

Current problems in dermatology, 1983

In order to explore the differential expression and evolutionary conservation of the genes encodi... more In order to explore the differential expression and evolutionary conservation of the genes encoding the cytoskeletal proteins for human epidermal cells, we have constructed a library of bacterial plasmids containing inserts of double-stranded cDNAs complementary to the mRNAs of cultured human epidermal cells. Cloned hybrid plasmids containing 45-95% of the sequences present in keratin, actin, and tubulin mRNAs were isolated and characterized. To identify the cDNAs encoding the four major keratins of human epidermal cells, the clones were initially screened for their ability to hybridize strongly with 32P-labeled cDNA prepared from unfractionated epidermal mRNA (about 30% keratin mRNA). Strongly hybridizing clones were further characterized by positive hybrid selection. Two distinct classes of clones were identified: One class hybridized specifically to the 56 and 58kd keratin mRNAs and one class hybridized specifically to the 46 and 50kd keratin mRNAs. Each class is encoded by a sep...

Journal of virology, 1986

The pol gene from a biologically active clone of the human T-cell lymphotropic virus type III pro... more The pol gene from a biologically active clone of the human T-cell lymphotropic virus type III provirus was inserted into a bacterial expression vector. The resulting gene fusion induced the formation of active reverse transcriptase that could be readily detected in extracts of bacterial cells. The activity exhibited the template and divalent cation requirements of the authentic enzyme. These constructs will be useful for safe and rapid analysis of potential inhibitors of this important enzyme.

Journal of virology, 1986

Portions of the pol gene of Moloney murine leukemia virus (MuLV) were expressed as fusion protein... more Portions of the pol gene of Moloney murine leukemia virus (MuLV) were expressed as fusion proteins in Escherichia coli, and the purified proteins were used to elicit antibodies in Escherichia coli, and the purified proteins were used to elicit antibodies in rabbits. The sera were used to examine the mature pol gene products contained in virion particles and identified the reverse transcriptase and a second protein, P46pol, encoded by the 3' portion of the gene. The P46 protein was not phosphorylated and was present at the same molar abundance as the reverse transcriptase. The sera were also used to detect the Pr200gag-pol intracellular precursor protein and to analyze its processing to the mature forms. The proteins formed by several Moloney MuLV mutants were analyzed. Further tests revealed cross-reactivity with Friend MuLV and feline leukemia virus proteins, but not with avian retrovirus proteins.

Journal of virology, 1990

The 3' terminus of the pol gene of Moloney murine leukemia virus encodes the integration (IN)... more The 3' terminus of the pol gene of Moloney murine leukemia virus encodes the integration (IN) protein, required for the establishment of the integrated provirus. A series of six linker insertion mutations and two single-base substitutions were generated within the region encoding the IN protein. Mutations were initially generated within an Escherichia coli plasmid expressing the IN protein, and the resulting variants were assayed for DNA-binding activity. Mutations which altered conserved cysteine residues within a potential DNA finger-binding motif resulted in lower or variable DNA binding, which appeared to be the result of variable protein folding. Upon renaturation, these proteins were able to nonspecifically bind DNA in a manner similar to that of the other mutant IN proteins and the parent. When reconstructed back into full-length virus, seven of the eight mutations were lethal. All mutants produced a stable IN protein in virions and mediated normal conversion of the retro...

Scientific reports, 2011

Transport of mRNAs to diverse neuronal locations via RNA granules serves an important function in... more Transport of mRNAs to diverse neuronal locations via RNA granules serves an important function in regulating protein synthesis within restricted sub-cellular domains. We recently detected the Huntington's disease protein huntingtin (Htt) in dendritic RNA granules; however, the functional significance of this localization is not known. Here we report that Htt and the huntingtin-associated protein 1 (HAP1) are co-localized with the microtubule motor proteins, the KIF5A kinesin and dynein, during dendritic transport of β-actin mRNA. Live cell imaging demonstrated that β-actin mRNA is associated with Htt, HAP1, and dynein intermediate chain in cultured neurons. Reduction in the levels of Htt, HAP1, KIF5A, and dynein heavy chain by lentiviral-based shRNAs resulted in a reduction in the transport of β-actin mRNA. These findings support a role for Htt in participating in the mRNA transport machinery that also contains HAP1, KIF5A, and dynein.

Cytometry Part A, 2012

The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase TrkB s... more The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase TrkB serve important regulatory roles for multiple aspects of the biology of neurons including cell death, survival, growth, differentiation, and plasticity. Regulation of the local availability of BDNF/TrkB at distinct subcellular domains such as soma, dendrites, axons, growth cones, nerve terminals, and spines appears to contribute to their specific functions. In view of the variance in size and shape of neurons and their compartments, previous quantitative studies of the BDNF/ TrkB protein and mRNA lacked a robust normalization procedure. To overcome this problem, we have established methods that use immunofluorescence detection of α-tubulin as a normalization factor for the quantitative analysis of protein and mRNA in primary rat cortical and striatal neurons in culture. The efficacy of this approach is demonstrated by studying the dynamic distribution of proteins and mRNA at different growth stages or conditions. Treatment of cultured neurons with KCl resulted in increased levels of TrkB protein, reduced levels of BDNF mRNA (composite of multiple transcripts) and a slight reduction in BDNF protein levels in the dendrites from the cortex. The KCl treatment also lowered the percentage of BDNF and TrkB proteins in the soma indicative of protein transport. Finally, analysis of the rat cortical and striatal neurons demonstrated comparable or even higher levels of BDNF/TrkB protein and BDNF mRNA in the neurons from the striatum. Thus, in contrast to previous observations made in vivo, striatal neurons are capable of synthesizing BDNF mRNA when cultured in growth media in vitro. The analytical approach presented here provides a detailed understanding of BDNF/TrkB levels in response to a variety of neuronal activities. Our methods could be used broadly, including applications in cell and tissue cytometry, to yield accurate quantitative data of gene expression in cellular and subcellular contexts.

Journal of Huntington's disease, Jan 29, 2016

The Huntington's disease (HD) protein huntingtin (Htt) plays a role in multiple cellular path... more The Huntington's disease (HD) protein huntingtin (Htt) plays a role in multiple cellular pathways. Deregulation of one or more of these pathways by the mutant Htt protein has been suggested to contribute to the disease pathogenesis. Our recent discovery-based proteomics studies have uncovered RNA binding proteins and translation factors associated with the endogenous Htt protein purified from mouse brains, suggesting a potential new role for Htt in RNA transport and translation. To investigate how Htt might affect RNA metabolism we set out to purify and analyze RNA associated with Htt. RNA was extracted from immunopurified Htt-containing protein complexes and analyzed by microarrays and RNA-Seq. Surprisingly, the most enriched mRNA that co-purified with Htt was Htt mRNA itself. The association of Htt protein and Htt mRNA was detected independent of intact ribosomes suggesting that it is not an RNA undergoing translation. Furthermore, we identified the recently reported mis-splic...

Proceedings of the National Academy of Sciences, 2008

Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansio... more Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansion of polyglutamines in the huntingtin (Htt) protein, whose cellular function remains controversial. To gain insight into Htt function, we purified epitope-tagged Htt and identified Argonaute as associated proteins. Colocalization studies demonstrated Htt and Ago2 to be present in P bodies, and depletion of Htt showed compromised RNA-mediated gene silencing. Mouse striatal cells expressing mutant Htt showed fewer P bodies and reduced reporter gene silencing activity compared with wild-type counterparts. These data suggest that the previously reported transcriptional deregulation in HD may be attributed in part to mutant Htt's role in post-transcriptional processes.

Molecular and Cellular Biology, 2010

The mammalian SWI/SNF chromatin-remodeling complex facilitates DNA access by transcription factor... more The mammalian SWI/SNF chromatin-remodeling complex facilitates DNA access by transcription factors and the transcription machinery. The characteristic member of human SWI/SNF-A is BAF250/ARID1, of which there are two isoforms, BAF250a/ARID1a and BAF250b/ARID1b. Here we report that BAF250b complexes purified from mammalian cells contain elongin C (Elo C), a BC box binding component of an E3 ubiquitin ligase. BAF250b was found to have a BC box motif, associate with Elo C in a BC box-dependent manner, and, together with cullin 2 and Roc1, assemble into an E3 ubiquitin ligase. The BAF250b BC box mutant protein was unstable in vivo and was autoubiquitinated in a manner similar to that for the VHL BC box mutants. The discovery that BAF250 is part of an E3 ubiquitin ligase adds an enzymatic function to the chromatin-remodeling complex SWI/SNF-A. The immunopurified BAF250b E3 ubiquitin ligase was found to target histone H2B at lysine 120 for monoubiquitination in vitro . To date, all H2B mo...

EMBO Journal, Dec 15, 1993

Regulation of transcription initiation by RNA polymerase II requires TFIID, a multisubunit comple... more Regulation of transcription initiation by RNA polymerase II requires TFIID, a multisubunit complex composed of the TATA binding protein (TBP) and at least seven tightly associated factors (TAFs). Some TAFs act as direct targets or coactivators for promoter-specific activators while others serve as interfaces for TAF-TAF interactions. Here, we report the molecular cloning, expression and characterization of Drosophila dTAFII60 and its human homolog, hTAFII70. Recombinant TAFII60/70 binds weakly to TBP and tightly to the largest subunit of TFIID, TAFII250. In the presence of TAFII60/70, TBP and TAFII250, a stable ternary complex is formed. Both the human and Drosophila proteins directly interact with another TFIID subunit, dTAFII40. Our findings reveal that Drosophila TAFII60 and human TAFII70 share a high degree of structural similarity and that their interactions with other subunits of TFIID are conserved.

Virology, 1989

An insertion mutation in the pal gene of Moloney murine leukemia virus (M-MuLV) was found to rend... more An insertion mutation in the pal gene of Moloney murine leukemia virus (M-MuLV) was found to render the virus temperature-sensitive for replication. A provirus containing a 12-bp insertion at the boundary between the reverse transcriptase (RT) and integrase (IN) domains induced the formation of mutant virions containing a partially processed RT-IN fusion protein. Some proteolytic processing to form mature RT and IN was observed at 32", but only aberrantly processed proteins were detected at 39". The uncleaved precursor was found to exhibit DNA polymerase activity, even though it could not support replication of the virus in viva at 39".

The Journal of biological chemistry, Jan 5, 1985

Expression of a region of the Moloney murine leukemia virus (M-MuLV) pol gene in Escherichia coli... more Expression of a region of the Moloney murine leukemia virus (M-MuLV) pol gene in Escherichia coli resulted in the synthesis of reverse transcriptase activity which could be detected in crude extracts. Construction of deletions at the 3' terminus of this gene resulted in a 4-fold increase in the level of the reverse transcriptase activity in the soluble fraction of crude lysates and yielded the high level production of a stable protein species of Mr = 71,000. Purification of this protein by column chromatography on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, and hydroxylapatite indicated that it was a multifunctional enzyme containing RNase H and reverse transcriptase activity. The Mr = 71,000 species had a sedimentation coefficient of 4.65 S by glycerol gradient centrifugation, indicating that the enzyme was a monomer. Using poly(A)+ mRNAs primed with oligo(dT), the enzyme synthesized double-stranded DNA copies between 1.3 and 9.9 kilobases in length. Synth...

Briefings in functional genomics, 2010

While initiation of transcription has attracted the most attention in the field of gene regulatio... more While initiation of transcription has attracted the most attention in the field of gene regulation, it has become clear that additional stages in the gene expression cascade including post-transcriptional events are under equally exquisite control. The seminal discovery that short RNAs (microRNA, small interfering RNA, Piwi-interacting RNA), play important roles in repressing gene expression has spurred a rush of new interest in post-transcriptional gene silencing mechanisms. The development of affinity tags and high-resolution tandem mass spectrometry (MS/MS) has greatly simplified the analysis of proteins that regulate gene expression. Further, the use of DNA microarrays and 'second generation' nucleic acid sequencing ('deep sequencing') technologies has facilitated the identification of their regulatory targets. These technological advancements mark a significant step towards a comprehensive understanding of gene regulatory networks. The purpose of this review is ...

Nature genetics, 2000

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding p... more At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration.

Molecular and cellular biology, 1998

TFIID is a multiprotein complex consisting of the TATA box binding protein and multiple tightly a... more TFIID is a multiprotein complex consisting of the TATA box binding protein and multiple tightly associated proteins (TAFIIs) that are required for transcription by selected activators. We previously reported cloning and partial characterization of human TAFII130 (hTAFII130). The central domain of hTAFII130 contains four glutamine-rich regions, designated Q1 to Q4, that are involved in interactions with the transcriptional activator Sp1. Mutational analysis has revealed specific regions within the glutamine-rich (Q1 to Q4) central region of hTAFII130 that are required for interaction with distinct activation domains. We tested amino- and carboxyl-terminal deletions of hTAFII130 for interaction with Sp1 activation domains A and B (Sp1A and Sp1B) and the N-terminal activation domain of CREB (CREB-N) by using the yeast two-hybrid system. Our results indicate that Sp1B interacts almost exclusively with the Q1 region of hTAFII130. In contrast, Sp1A makes multiple contacts with Q1 to Q4 of...

Journal of virology, 1997

The human cytomegalovirus (HCMV) major immediate-early (IE) proteins IEP86 (IE2(579aa)) and IEP72... more The human cytomegalovirus (HCMV) major immediate-early (IE) proteins IEP86 (IE2(579aa)) and IEP72 (IE1(491aa)) can transcriptionally activate a variety of simple promoters containing a TATA element and one upstream transcription factor binding site. In our previous studies, transcriptional activation was shown to correlate with IEP86 binding to both the TATA-box binding protein (TBP) and the transcription factor bound upstream. IEP72 often synergistically affects the activation by IEP86, although it has not previously been shown to directly interact in vitro with IEP86, TBP, or transcription factors (e.g., Sp1 and Tef-1) bound by IEP86. We report biochemical and genetic evidence suggesting that the major IE proteins may perform a function similar to that of the TBP-associated factors (TAFs) which make up TFIID. Consistent with this model, we found that the major IE proteins interact with a number of TAFs. In vitro, IEP86 bound with drosophila TAF(II)110 (dTAF(II)110) and human TAF(I...

The EMBO journal, Jan 15, 1993

Regulation of transcription initiation by RNA polymerase II requires TFIID, a multisubunit comple... more Regulation of transcription initiation by RNA polymerase II requires TFIID, a multisubunit complex composed of the TATA binding protein (TBP) and at least seven tightly associated factors (TAFs). Some TAFs act as direct targets or coactivators for promoter-specific activators while others serve as interfaces for TAF-TAF interactions. Here, we report the molecular cloning, expression and characterization of Drosophila dTAFII60 and its human homolog, hTAFII70. Recombinant TAFII60/70 binds weakly to TBP and tightly to the largest subunit of TFIID, TAFII250. In the presence of TAFII60/70, TBP and TAFII250, a stable ternary complex is formed. Both the human and Drosophila proteins directly interact with another TFIID subunit, dTAFII40. Our findings reveal that Drosophila TAFII60 and human TAFII70 share a high degree of structural similarity and that their interactions with other subunits of TFIID are conserved.

Current problems in dermatology, 1983

In order to explore the differential expression and evolutionary conservation of the genes encodi... more In order to explore the differential expression and evolutionary conservation of the genes encoding the cytoskeletal proteins for human epidermal cells, we have constructed a library of bacterial plasmids containing inserts of double-stranded cDNAs complementary to the mRNAs of cultured human epidermal cells. Cloned hybrid plasmids containing 45-95% of the sequences present in keratin, actin, and tubulin mRNAs were isolated and characterized. To identify the cDNAs encoding the four major keratins of human epidermal cells, the clones were initially screened for their ability to hybridize strongly with 32P-labeled cDNA prepared from unfractionated epidermal mRNA (about 30% keratin mRNA). Strongly hybridizing clones were further characterized by positive hybrid selection. Two distinct classes of clones were identified: One class hybridized specifically to the 56 and 58kd keratin mRNAs and one class hybridized specifically to the 46 and 50kd keratin mRNAs. Each class is encoded by a sep...

Journal of virology, 1986

The pol gene from a biologically active clone of the human T-cell lymphotropic virus type III pro... more The pol gene from a biologically active clone of the human T-cell lymphotropic virus type III provirus was inserted into a bacterial expression vector. The resulting gene fusion induced the formation of active reverse transcriptase that could be readily detected in extracts of bacterial cells. The activity exhibited the template and divalent cation requirements of the authentic enzyme. These constructs will be useful for safe and rapid analysis of potential inhibitors of this important enzyme.

Journal of virology, 1986

Portions of the pol gene of Moloney murine leukemia virus (MuLV) were expressed as fusion protein... more Portions of the pol gene of Moloney murine leukemia virus (MuLV) were expressed as fusion proteins in Escherichia coli, and the purified proteins were used to elicit antibodies in Escherichia coli, and the purified proteins were used to elicit antibodies in rabbits. The sera were used to examine the mature pol gene products contained in virion particles and identified the reverse transcriptase and a second protein, P46pol, encoded by the 3' portion of the gene. The P46 protein was not phosphorylated and was present at the same molar abundance as the reverse transcriptase. The sera were also used to detect the Pr200gag-pol intracellular precursor protein and to analyze its processing to the mature forms. The proteins formed by several Moloney MuLV mutants were analyzed. Further tests revealed cross-reactivity with Friend MuLV and feline leukemia virus proteins, but not with avian retrovirus proteins.

Journal of virology, 1990

The 3' terminus of the pol gene of Moloney murine leukemia virus encodes the integration (IN)... more The 3' terminus of the pol gene of Moloney murine leukemia virus encodes the integration (IN) protein, required for the establishment of the integrated provirus. A series of six linker insertion mutations and two single-base substitutions were generated within the region encoding the IN protein. Mutations were initially generated within an Escherichia coli plasmid expressing the IN protein, and the resulting variants were assayed for DNA-binding activity. Mutations which altered conserved cysteine residues within a potential DNA finger-binding motif resulted in lower or variable DNA binding, which appeared to be the result of variable protein folding. Upon renaturation, these proteins were able to nonspecifically bind DNA in a manner similar to that of the other mutant IN proteins and the parent. When reconstructed back into full-length virus, seven of the eight mutations were lethal. All mutants produced a stable IN protein in virions and mediated normal conversion of the retro...