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BBA - Gene Regulatory Mechanisms (v.1789, #11-12)

Editorial Board (pp. i).

A-form DNA structure is a determinant of transcript levels from the Xenopus gata2 promoter in embryos by Katrina J. Llewellyn; Peter D. Cary; James A. McClellan; Matthew J. Guille; Garry P. Scarlett (pp. 675-680).

We have previously shown that a critical region of the gata2 promoter contains an inverted CCAAT box and adopts a partial A-form DNA structure in vitro. At gastrula stages of development transcription requires binding of CBTF (CCAAT box transcription factor), a multi-subunit transcription factor, to this region. Xilf3 is one component of CBTF and the double stranded RNA binding domains (dsRBDs) of Xilf3 must be active for both binding to, and transcription from, this promoter. Here we determine the contribution of DNA sequence and structure at the gata2 promoter to transcriptional activity. In all the constructs we tested a CCAAT box was a requirement for full activity. However, base substitutions that increase B-form structure propensity in the sequences flanking the CCAAT box are equally able to decrease activity even if a CCAAT box is present. In contrast, mutations that maintain A-form propensity in these regions also maintain, or increase, transcription factor binding and transcriptional activity. We propose a two-component model for the interaction of CBTF with the gata2 promoter, requiring both a CCAAT sequence and flanking A-form DNA structures. These results support a novel role for dsRBDs in transcriptional regulation and suggest a function for A-form DNA in vivo.

Keywords: Transcription; A-Form DNA; Xilf3; gata2

Transcriptional regulation of neutral sphingomyelinase 2 gene expression of a human breast cancer cell line, MCF-7, induced by the anti-cancer drug, daunorubicin by Hiromi Ito; Masashi Murakami; Ayako Furuhata; Siqiang Gao; Kayo Yoshida; Sayaka Sobue; Kazumi Hagiwara; Akira Takagi; Tetsuhito Kojima; Motoshi Suzuki; Yoshiko Banno; Kouji Tanaka; Keiko Tamiya-Koizumi; Mamoru Kyogashima; Yoshinori Nozawa; Takashi Murate (pp. 681-690).

Mg²⁺-dependent neutral SMases (NSMases) have emerged as prime candidates for stress-induced ceramide production. Among isoforms identified, previous reports have suggested the importance of NSMase2. However, its activation mechanism has not been precisely reported. Here, we analyzed the mechanism of NSMase2 gene expression by the anti-cancer drug, daunorubicin (DA). DA increased cellular ceramides (C16, C18 and C24) and NSMase activity of a human breast cancer cell line, MCF-7. DA remarkably increased the NSMase2 message and protein, whereas little change in NSMase1 and NSMase3 mRNAs and only a mild increase in acid SMase mRNA were observed. Overexpression and a knock down of NSMase2 indicated that NSMase2 played a role in DA-induced cell death. NSMase2 promoter analysis revealed that three Sp1 motifs located between −148 and −42bp upstream of the first exon were important in basic as well as in DA-induced promoter activity. Consistently, luciferase vectors containing three consensus Sp1-motifs but not its mutated form showed DA-induced transcriptional activation. DA-treated MCF-7 showed increased Sp3 protein. In SL2 cells lacking Sp family proteins, both Sp1 and Sp3 overexpression increased NSMase promoter activity. Increased binding of Sp family proteins by DA to three Sp1 motifs was shown by electrophoresis mobility shift and ChIP assays.

Keywords: Abbreviations; SMase; sphingomyelinase; SM; sphingomyelin; EMSA; electrophoresis mobility shift assay; ChIP; chromatin immunoprecipitation; DA; daunorubicin; Sp; specificity proteinNSMase2; Daunorubicin; Ceramide; Promoter analysis; EMSA; ChIP; Sp family protein

The 19S proteasome positively regulates histone methylation at cytokine inducible genes by Olivia I. Koues; R. Kyle Dudley; Ninad T. Mehta; Susanna F. Greer (pp. 691-701).

Studies indicate that the 19S proteasome functions in the epigenetic regulation of transcription. We have shown that as in yeast, components of the 19S proteasome are crucial for regulating inducible histone acetylation events in mammalian cells. The 19S ATPase Sug1 binds to histone acetyltransferases and to acetylated histone H3 and, in the absence of Sug1, histone H3 acetylation is dramatically decreased at mammalian promoters. Research in yeast further indicates that the ortholog of Sug1, Rpt6, is a link between ubiquitination of histone H2B and H3 lysine 4 trimethylation (H3K4me3). To characterize the role that the 19S proteasome plays in regulating additional activating modifications, we examined the methylation and ubiquitination status of histones at inducible mammalian genes. We find that Sug1 is crucial for regulating histone H3K4me3 and H3R17me2 at the cytokine inducible MHC-II and CIITA promoters. In the absence of Sug1, histone H3K4me3 and H3R17me2 are dramatically decreased, but the loss of Sug1 has no significant effect on H3K36me3 or H2BK120ub. Our observation that a subunit of hCompass interacts with additional activating histone modifying enzymes, but fails to bind the CIITA promoter in the absence of Sug1, strongly implicates Sug1 in recruiting enzyme complexes responsible for initiating mammalian transcription.

Keywords: Sug1; 26S proteasome; Transcriptional regulation; Epigenetic; Compass; WDR5

Crlz1 activates transcription by mobilizing cytoplasmic CBFβ into the nucleus by Sung-Kyun Park; Jung-Hyun Lim; Chang-Joong Kang (pp. 702-708).

Transcriptional function of a novel Crlz1 protein was examined by using the CBF site-containing IgJ enhancer, because it was originally cloned due to its ability to bind CBFβ, a subunit of CBF heterodimer, of which Runx is the other subunit. In a cotransfection experiment, Crlz1 was shown to increase the IgJ enhancer activity due to its CBF sites, as verified by both the absence of Crlz1 effect on the CBF-site mutated IgJ enhancer and the presence of transcriptional synergy between Crlz1 and CBFβ. Most significantly, the cytoplasmic CBFβ was shown to be mobilized into the nucleus when it was coexpressed with the nuclear Crlz1. This mobilized nuclear CBFβ could then heterodimerize with the nuclear Runx to bind to its target DNA site with a high affinity. Furthermore, in our coimmunoprecipitation and chromatin immunoprecipitation experiments, Crlz1 was found to be bound to the resulting CBF heterodimer in a form of ternary complex and to remain in that ternary complex even when CBF bound to its target DNA site such as IgJ enhancer.

Keywords: Crlz1; CBFβ nuclear mobilization; Runx-CBFβ heterodimerization

Interaction of intestinal and pancreatic transcription factors in the regulation of CFTR gene expression by Victoria A. McCarthy; Christopher J. Ott; Marios Phylactides; Ann Harris (pp. 709-718).

The tissue-specific regulation of the cystic fibrosis transmembrane conductance regulator gene ( CFTR) is coordinated by intronic and extragenic cis-acting elements that influence its transcriptional activity. The promoter apparently lacks sequences to drive cell type-specific expression. We previously identified a number of intronic elements that were associated with DNase I hypersensitive sites (DHS) and bound the hepatocyte nuclear factor 1 (HNF1) transcription factor. Moreover, we demonstrated the likely involvement of HNF1 in the regulation of CFTR expression in vivo. Here we investigate DHS in introns 16 and 17a of the CFTR gene, which are evident in intestinal and pancreatic cell lines, and determine the transcription factors that interact with these sites. Of particular interest were factors known to interact with HNF1 in coordinated expression of genes in the gastrointestinal tract. We demonstrate that though sequences within these DHS bind HNF1, CDX2, and PBX1 in vitro, only PBX1 show a robust in vivo interaction. These data contribute to our understanding of the complexity of cell-type-specific CFTR regulatory mechanisms.

Keywords: CFTR regulation; DHS; Cis-acting element; HNF1; CDX2; PBX1

Biotin is not a natural histone modification by Shannon Healy; Beatriz Perez-Cadahia; Dongxin Jia; Megan K. McDonald; James R. Davie; Roy A. Gravel (pp. 719-733).

In addition to its role as the cofactor of biotin-dependent carboxylases, biotin has been demonstrated to have a role in cellular processes including transcription and gene silencing. Histones have been proposed to be modified by biotin in a site-specific manner, providing a pathway by which biotin acts as a regulatory molecule for gene expression. However, there is uncertainty whether biotin attachment to histones in vitro can be extrapolated to biotin as a native histone modification. We critically examined a number of methods used to detect biotin attachment on histones, including [³H]-biotin uptake, Western blot analysis of histones, and mass spectrometry of affinity purified histone fragments with the objective of determining if the in vivo occurrence of histone biotinylation could be conclusively established. We found for each of these methods that, while biotin could be readily detected on native carboxylases or histones biotinylated in vitro, biotin attachment on native histones could not be detected in cell cultures from various sources. We conclude that biotin is absent in native histones to a sensitivity of at least one part per 100,000, suggesting that the regulatory impact of biotin on gene expression must be through alternate mechanisms.

Keywords: Abbreviations; ACC; acetyl-CoA caroboxylase; BSA; bovine serum albumin; BTD; biotinidase; bio-5′-AMP; biotinyl-5′-AMP; HCS; holocarboxylase synthetase; HRP; horseradish peroxidase; Lys; lysine; MALDI; matrix-assisted laser desorption/ionization; MS; mass spectrometry; MCC; β-methylcrotonyl-CoA carboxylase; MW; molecular weight; m/z; mass/charge; PBS; phosphate-buffered saline solution; PC; pyruvate carboxylase; PCC; propionyl-CoA carboxylaseBiotin; Histones; Post-translational modification; Immunoblot; Mass spectrometry

Activation of testicular orphan receptor 4 by fatty acids by Nien-Pei Tsai; Mostaqul Huq; Pawan Gupta; Keiko Yamamoto; Hiroyuki Kagechika; Li-Na Wei (pp. 734-740).

Nuclear receptors can be activated by chemicals, metabolites, hormones or environmental compounds to regulate gene expression. Bioassay-guided screening of mouse tissue extracts found that natural fatty acids of a certain carbon length and level of unsaturation could activate the mouse orphan nuclear receptor, testicular orphan receptor 4 (TR4). Subsequent experiments focused on γ-linoleic acid, a compound identified during screening of mouse tissues that exerts regulatory activity in TR4 transactivation assays. γ-linoleic acid positively modulates TR4 activity to promote the expression of downstream genes such as apolipoprotein E (ApoE) and phosphoenolpyruvate carboxykinase, and to activate a reporter carrying direct repeat 1 from the ApoE promoter. It also induced the interaction of TR4 with transcription coregulators such as RIP140 and PCAF. Comparisons of transactivation by TR4 and the metabolism-related peroxisome proliferator-activated nuclear receptors indicate that γ-linoleic acid regulation is specific to TR4. The data suggest that TR4 might exert its physiological function by sensing certain lipids. Identifying these compounds could be useful for examining the physiological pathways in which TR4 and its target genes are involved.

Keywords: TR4; Linoleic acid; ApoE; PEPCK

Determination of the core promoter regions of the Saccharomyces cerevisiae RPS3 gene by Yoo Jin Joo; Jin-ha Kim; Joung Hee Baek; Ki Moon Seong; Jae Yung Lee; Joon Kim (pp. 741-750).

Ribosomal protein genes (RPG), which are scattered throughout the genomes of all eukaryotes, are subjected to coordinated expression. In yeast, the expression of RPGs is highly regulated, mainly at the transcriptional level. Recent research has found that many ribosomal proteins (RPs) function in multiple processes in addition to protein synthesis. Therefore, detailed knowledge of promoter architecture as well as gene regulation is important in understanding the multiple cellular processes mediated by RPGs. In this study, we investigated the functional architecture of the yeast RPS3 promoter and identified many putative cis-elements. Using β-galactosidase reporter analysis and EMSA, the core promoter of RPS3 containing UAS_rpg and T-rich regions was corroborated. Moreover, the promoter occupancy of RPS3 by three transcription factors was confirmed. Taken together, our results further the current understanding of the promoter architecture and trans-elements of the Saccharomyces cerevisiae RPS3 gene.

Keywords: Rap1p; UAS; _rpg; T-rich region; Transcription factor; Ribosomal protein gene

The maize HMGA protein is localized to the nucleolus and can be acetylated in vitro at its globular domain, and phosphorylation by CDK reduces its binding activity to AT-rich DNA by Jing Zhao; Laju K. Paul; Gideon Grafi (pp. 751-757).

The high mobility group (HMG) proteins are nonhistone chromosomal proteins that participate in diverse nuclear activities including chromatin structure and gene regulation. We previously studied the biochemistry of the maize HMGA protein and its role in transcriptional regulation during maize endosperm development. Here, we extended our study and showed that a strong binding of ZmHMGA to AT-rich DNA requires at least three AT-hook motifs; two motifs showed a significant reduction whereas a single motif was not sufficient for binding. CDK phosphorylation sites situated between AT-hook3 and AT-hook4 were strongly phosphorylated by a SUC1-associated kinase; no in vitro phosphorylation is evident for the AtHMGA protein. Phosphorylation of ZmHMGA reduced its binding to AT-rich DNA in vitro. The maize HMGA protein fused to GFP was localized in the nucleus of transgenic Arabidopsis plants tending to concentrate within the nucleolus. Localization to the nucleolus was conferred by the C-terminal portion of the protein containing the AT-hooks. ZmHMGA was acetylated in vitro on its N-terminal globular domain by the human PCAF acetyltransferase. Our results suggest that ZmHMGA participates in nucleolar function and that its role may be regulated posttranslationally by phosphorylation and acetylation.

Keywords: Abbreviations; HMG; high mobility group; CDK; cyclin-dependent kinaseHigh mobility group A; AT-hook; Posttranslational modification; Acetylation; Phosphorylation; Nucleolus; Maize; Arabidopsis

An inhibitory role of p53 via NF-κB element on the cyclin D1 gene under heat shock by Zhiyi Guo; Jinshan Wang; Jun Yang; Ning-hua Wu; Ye Zhang; Yu-fei Shen (pp. 758-762).

Little is known about the mechanisms underlying heat shock-mediated inhibition of cyclin D1 transcription. Here, we report that NF-κB site-mediated cyclin D1 transcription is inhibited by heat shock. The mRNA level of cyclin D1 decreased under heat shock (40–60%). This inhibition of transcription is promoter activity dependent and is mediated by the proximal NF-κB site. However, P65 overexpression did not influence the heat-inducible inhibitory pattern and heat shock did not significantly change the binding activity of p65. P53 can inhibit cyclin D1 promoter activity via an NF-κB site-dependent manner and its binding activity increased after heat shock. Importantly, p53 overexpression can prevent cyclin D1 promoter activation by p65. Therefore, we can deduce that p53 inhibits promoter activity under heat shock. These results reveal that the mechanism of heat shock-mediated inhibition of cyclin D1 transcription involves an NF-κB site. The data presented provide a new insight into the underlying heat shock inhibition of gene transcription.

Keywords: cyclin D1; Heat shock; Promoter; NF-κB; p53

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BBA - Gene Regulatory Mechanisms (v.1789, #11-12)