Cheol Kyu Hwang

Three-amino acid Extension Loop Homeodomain Proteins Meis2 and TGIF Differentially Regulate Transcription

Three-amino acid extension loop (TALE) homeobox proteins are highly conserved transcription regulators. We report that two members of this family, Meis2 and TGIF, which frequently have overlapping consensus binding sites on complementary DNA strands in opposite orientations, can function competitively. For example, in theD1A gene, which encodes the predominant dopamine receptor in the striatum, Meis2 and TGIF bind to the activator sequence ACT (−1174 to −1154) and regulate transcription differentially in a cell type-specific manner. Among the five cloned splice variants of Meis2, isoforms Meis2a–d activate theD1A promoter in most cell types tested, whereas TGIF competes with Meis2 binding to DNA and represses Meis2-induced transcription activation. Consequently, Meis2 cannot activate theD1A promoter in a cell that has abundant TGIF expression. The Meis2 message is highly co-localized with the D1A message in adult striatal neurons, whereas TGIF is barely detectable in the adult brain. Our observations provide in vitro and in vivo evidence that Meis2 and TGIF differentially regulate their target genes. Thus, the delicate ratio between Meis2 and TGIF expression in a given cell type determines the cell-specific expression of theD1A gene. We also found that splice variant Meis2e, which has a truncated homeodomain, cannot bind to theD1A ACT sequence or activate transcription. However, Meis2e is an effective dominant negative regulator by blocking Meis2d-induced transcription activation. Thus, truncated homeoproteins with no DNA binding domains can have important regulatory functions.

Dopamine receptor regulating factor, DRRF: A zinc finger transcription factor

Dopamine receptor genes are under complex transcription control, determining their unique regional distribution in the brain. We describe here a zinc finger type transcription factor, designated dopamine receptor regulating factor (DRRF), which binds to GC and GT boxes in the D1A and D2 dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Consequently, DRRF can modulate the activity of these dopamine receptor promoters. Highest DRRF mRNA levels are found in brain with a specific regional distribution including olfactory bulb and tubercle, nucleus accumbens, striatum, hippocampus, amygdala, and frontal cortex. Many of these brain regions also express abundant levels of various dopamine receptors. In vivo, DRRF itself can be regulated by manipulations of dopaminergic transmission. Mice treated with drugs that increase extracellular striatal dopamine levels (cocaine), block dopamine receptors (haloperidol), or destroy dopamine terminals (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) show significant alterations in DRRF mRNA. The latter observations provide a basis for dopamine receptor regulation after these manipulations. We conclude that DRRF is important for modulating dopaminergic transmission in the brain.

A Proteomics Approach for Identification of Single Strand DNA-binding Proteins Involved in Transcriptional Regulation of Mouse μ Opioid Receptor Gene

The pharmacological actions of morphine and morphine-like drugs such as heroin are mediated primarily through the μ opioid receptor. Previously a single strand DNA element of the mouse μ opioid receptor gene (Oprm1) proximal promoter was found to be important for regulating Oprm1 in neuronal cells. To identify proteins binding to the single strand DNA element as potential regulators for Oprm1, affinity column chromatography with the single strand DNA element was performed using neuroblastoma NS20Y cells followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified five poly(C)-binding proteins: heterogeneous nuclear ribonucleoprotein (hnRNP) K, α-complex proteins (αCP) αCP1, αCP2, αCP2-KL, and αCP3. Binding of these proteins to the single strand DNA element of Oprm1 was sequence-specific as confirmed by supershift assays. In cotransfection studies, hnRNP K, αCP1, αCP2, and αCP2-KL activated the Oprm1 promoter activity, whereas αCP3 acted as a repressor. Ectopic expression of hnRNP K, αCP1, αCP2, and αCP2-KL also led to activation of the endogenous Oprm1 transcripts, and αCP3 repressed endogenous Oprm1 transcripts. We demonstrate novel roles as transcriptional regulators in Oprm1 regulation for hnRNP K and αCP binding to the single strand DNA element.

Characterization of the 5′ flanking region of the rat D3 dopamine receptor gene

The D3 dopamine receptor has a restricted regional distribution in brain and is regulated by dopaminergic agents. Additionally, the D3 gene is implicated in the pathogenesis of several neuropsychiatric disorders or in their response to pharmacological agents. Elucidating its transcription control mechanisms is therefore of interest in order to explain these biological features of the D3 gene. In this study, the 5′ flanking region of the rat D3 gene was characterized by isolating the 5′ end of its cDNA as well as 4.6 kb of genomic sequence. Analysis of this region revealed the presence of two new exons 196‐bp and 120‐bp long, separated by an 855‐bp intron, located several kilobases upstream of the previously published coding exons. Thus, current evidence indicates that the rat D3 gene is organized into eight exons. Transcription initiation site was determined by primer extension analysis and repeated rounds of 5′ RACE and was found to localize at a pyrimidine‐rich consensus ‘initiator’ sequence, similar to the rat D2 gene. The D3 promoter lacks TATA or CAAT boxes but unlike that of other dopamine receptor genes has only 52% GC content. Functional analysis of D3 promoter deletion mutants fused to a reporter gene in TE671 cells, which endogenously express this gene, revealed strong transcriptional activity localized within 36 nucleotides upstream of transcription start site, and a potent silencer between bases −37 and −537. The D3 promoter is inactive in C6 and COS7 cells. We conclude that the D3 gene, similar to the closely related D2 gene, is transcribed from a tissue specific promoter which is under intense negative control.

Developmental expression of the zinc finger transcription factor DRRF (dopamine receptor regulating factor)

Dopamine receptor regulating factor (DRRF) is a novel transcription factor with unique anatomical distribution and functional properties, suggesting its importance in regulating dopaminergic neurotransmission. To gain insight into the in vivo function of this factor during embryogenesis, we studied its distribution at embryonic days E8-E16 in the mouse using in situ hybridization. DRRF mRNA is expressed uniquely during development at all time points tested with high levels observed at E12, E14 and E16 in various tissues. DRRF expression is also found in particular brain regions, such as the neopallial cortex, olfactory lobe and corpus striatum. This pattern of DRRF distribution during embryogenesis overlaps with that found in the adult brain, and with the expression profile of dopamine receptors both in the adult and during development.

Characterization of the 5'flanking region of the rat D(3) dopamine receptor gene.

The D3 dopamine receptor has a restricted regional distribution in brain and is regulated by dopaminergic agents. Additionally, the D3 gene is implicated in the pathogenesis of several neuropsychiatric disorders or in their response to pharmacological agents. Elucidating its transcription control mechanisms is therefore of interest in order to explain these biological features of the D3 gene. In this study, the 5′ flanking region of the rat D3 gene was characterized by isolating the 5′ end of its cDNA as well as 4.6 kb of genomic sequence. Analysis of this region revealed the presence of two new exons 196‐bp and 120‐bp long, separated by an 855‐bp intron, located several kilobases upstream of the previously published coding exons. Thus, current evidence indicates that the rat D3 gene is organized into eight exons. Transcription initiation site was determined by primer extension analysis and repeated rounds of 5′ RACE and was found to localize at a pyrimidine‐rich consensus ‘initiator’ sequence, similar to the rat D2 gene. The D3 promoter lacks TATA or CAAT boxes but unlike that of other dopamine receptor genes has only 52% GC content. Functional analysis of D3 promoter deletion mutants fused to a reporter gene in TE671 cells, which endogenously express this gene, revealed strong transcriptional activity localized within 36 nucleotides upstream of transcription start site, and a potent silencer between bases −37 and −537. The D3 promoter is inactive in C6 and COS7 cells. We conclude that the D3 gene, similar to the closely related D2 gene, is transcribed from a tissue specific promoter which is under intense negative control.

Characterization of the 5′ flanking region of the rat D3 dopamine receptor gene: The 5' flanking region of the rat D 3 dopamine receptor gene

The D3 dopamine receptor has a restricted regional distribution in brain and is regulated by dopaminergic agents. Additionally, the D3 gene is implicated in the pathogenesis of several neuropsychiatric disorders or in their response to pharmacological agents. Elucidating its transcription control mechanisms is therefore of interest in order to explain these biological features of the D3 gene. In this study, the 5′ flanking region of the rat D3 gene was characterized by isolating the 5′ end of its cDNA as well as 4.6 kb of genomic sequence. Analysis of this region revealed the presence of two new exons 196‐bp and 120‐bp long, separated by an 855‐bp intron, located several kilobases upstream of the previously published coding exons. Thus, current evidence indicates that the rat D3 gene is organized into eight exons. Transcription initiation site was determined by primer extension analysis and repeated rounds of 5′ RACE and was found to localize at a pyrimidine‐rich consensus ‘initiator’ sequence, similar to the rat D2 gene. The D3 promoter lacks TATA or CAAT boxes but unlike that of other dopamine receptor genes has only 52% GC content. Functional analysis of D3 promoter deletion mutants fused to a reporter gene in TE671 cells, which endogenously express this gene, revealed strong transcriptional activity localized within 36 nucleotides upstream of transcription start site, and a potent silencer between bases −37 and −537. The D3 promoter is inactive in C6 and COS7 cells. We conclude that the D3 gene, similar to the closely related D2 gene, is transcribed from a tissue specific promoter which is under intense negative control.

p38 Mitogen-activated protein kinase and PI3-kinase are involved in up-regulation of mu opioid receptor transcription induced by cycloheximide: Control of MOR transcription by PI3- and p38 MAP kinases

J. Neurochem. (2011) 116, 1077–1087.

p38 MAP kinase and PI3-kinase are involved in upregulation of mu opioid receptor transcription induced by cycloheximide

J. Neurochem. (2011) 116, 1077–1087.