Alternative Promoter Utilization Research Articles

Tissue-selective alternate promoters guide NLRP6 expression.

The pryin domain (PYD) domain is involved in protein interactions that lead to assembly of immune-sensing complexes such as inflammasomes. The repertoire of PYD-containing genes expressed by a cell type arms tissues with responses against a range of stimuli. The transcriptional regulation of the PYD gene family however is incompletely understood. Alternative promoter utilization was identified as a mechanism regulating the tissue distribution of human PYD gene family members, including NLRP6 that is translationally silenced outside of intestinal tissue. Results show that alternative transcriptional promoters mediate NLRP6 silencing in mice and humans, despite no upstream genomic synteny. Human NLRP6 contains an internal alternative promoter within exon 2 of the PYD, resulting in a truncated mRNA in nonintestinal tissue. In mice, a proximal promoter was used that expanded the 5' leader sequence restricting nuclear export and abolishing translational efficiency. Nlrp6 was dispensable in disease models targeting the kidney, which expresses noncanonical isoforms. Thus, alternative promoter use is a critical mechanism not just for isoform modulation but for determining expression profile and function of PYD family members.

Epigenomic promoter alterations predict for benefit from immune checkpoint inhibition in metastatic gastric cancer

BackgroundUtilization of alternative transcription start sites through alterations in epigenetic promoter regions causes reduced expression of immunogenic N-terminal peptides, which may facilitate immune evasion in early gastric cancer. We hypothesized that tumors with high alternate promoter utilization would be resistant to immune checkpoint inhibition in metastatic gastric cancer.Patients and methodsTwo cohorts of patients with metastatic gastric cancer treated with immunotherapy were analyzed. The first cohort (N = 24) included patients treated with either nivolumab or pembrolizumab. Alternate promoter utilization was measured using the NanoString® (NanoString Technologies, Seattle, WA, USA) platform on archival tissue samples. The second cohort was a phase II clinical trial of patients uniformly treated with pembrolizumab (N = 37). Fresh tumor biopsies were obtained, and transcriptomic analysis was carried out on RNAseq data. Alternate promoter utilization was correlated to T-cell cytolytic activity, objective response rate and survival.ResultsIn the first cohort 8 of 24 (33%) tumors were identified to have high alternate promoter utilization (APhigh), and this was used to define the APhigh tertile of the second cohort (13 APhigh of 37). APhigh tumors exhibited decreased markers of T-cell cytolytic activity and lower response rates (8% versus 42%, P = 0.03). Median progression-free survival was lower in the APhigh group (55 versus 180 days, P = 0.0076). In multivariate analysis, alternative promoter utilization was an independent predictor of immunotherapy survival [hazard ratio 0.29, 95% confidence interval 0.099–0.85, P = 0.024). Analyzing tumoral evolution through paired pre-treatment and post-treatment biopsies, we observed consistent shifts in alternative promoter utilization rate associated with clinical response.ConclusionA substantial proportion of metastatic gastric cancers utilize alternate promoters as a mechanism of immune evasion, and these tumors may be resistant to anti-PD1 immune checkpoint inhibition. Alternate promoter utilization is thus a potential mechanism of resistance to immune checkpoint inhibition, and a novel predictive biomarker for immunotherapy.Trial RegistrationClinicalTrials.gov Identifier: NCT#02589496

I09 Adenosine deaminae (ADAR1) acting on double-stranded RNA and protein kinase PKR dependent on dsRNA as opposing modulators of antiviral innate immunity

Double-stranded RNA (dsRNA) plays a central role in antiviral innate immunity, both as a trigger for the production of interferon (IFN) and also in the actions of IFN. Adenosine deaminases acting on RNA (ADAR) catalyze the C-6 deamination of adenosine (A) to generate inosine (I) in dsRNAs. Because I hydrogen bonds as G with C, not as A with U, A-to-I editing can lead to genetic recoding of RNAs and destabilization of dsRNA structures. The Adar1 gene is IFN inducible and encodes two size forms of ADAR1 protein through alternative promoter utilization; an IFN inducible promoter specifies a p150 protein that is cytoplasmic and nuclear, whereas the constitutive promoters specify a smaller constitutively expressed p110 nuclear protein. Protein kinase PKR also is IFN inducible, is activated by dsRNA, and inhibits translation through eIF-2alpha factor phosphorylation. The roles of ADAR1 p150 and p110, and PKR, during virus replication were examined using human cell clones made stably deficient in ADAR1 or PKR by an RNA interference strategy, and mouse MEF cells genetically deficient in either ADAR1 or 2. Results obtained with wild-type measles virus (MV) and mutants deleted for either C or V expression revealed important roles for both ADAR1 and PKR as modulators of IFN induction and the host response to infection. The effects of ADAR1 deficiency on MV growth, IFN beta gene induction, and virus-induced cytopathic effect generally were opposite to the effects observed for PKR deficiency. The findings indicate that ADAR1 behaves as an anti-apoptotic host factor that, in some instances, is pro-viral. The anti-apoptotic and cell protective effects of ADAR1 correlated with suppression of activation of pro-apoptotic activities exemplified by PKR. Results obtained with mouse cells genetically deficient in adar1 likewise showed a protective role of ADAR1 protein isoforms against virus-induced cytopathic effects. (Supported by NIAID, NIH).

Abstract 270: The TWIST1-microRNA-22 axis represses estrogen receptor alpha expression in breast cancer

Abstract Background: TWIST1 (Twist) is a master regulator of embryonic cell fate specification and differentiation. It mediates its transrepressive and transactivating functions by binding to consensus E-box sequences within promoters. Importantly, ectopic expression of Twist in breast epithelial tissue is an initiator of breast cancer. Estrogen and estrogen receptor (ER) expression play a crucial role in breast cancer. ER expression is regulated by methylation, chromatin inactivation, alternative promoter utilization, mutations, as well as cis and trans-acting elements. MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression. We show here that Twist binds to E-boxes in the promoter of the microRNA miR-22 and upregulates its expression in breast cancer. Furthermore, the dysregulated miR-22 binds to the 3’-UTR of ER and downregulates its expression leading to an ER negative state. Methods: We isolated miRNA from breast cancer cells and assayed for miR-22 expression by qRT-PCR. A miR-22 construct was transfected into breast cancer cells to force expression of miR-22 and analyze effects on ER. Inhibition studies were carried out using targeted knockdowns of miR-22 and analyzed by qRT-PCR. Fine mapping of the miR-22 genomic structure was performed by RACE. The miR-22 promoter containing three E-boxes was cloned and used in dual luciferase assays to study Twist binding and miR-22 activation. Chromatin immunoprecipitation assays were carried out to confirm Twist binding. The 3’-UTR of the ER was cloned into the pMIR-Report vector and the construct used to study miR-22 binding. Functionality of miR-22 binding to the ER 3’-UTR leading to loss of ER expression was confirmed by immunoblotting. Results: We found that miR-22 was highly expressed in cell lines expressing Twist and that ER expression was inversely proportional to miR-22 in all cancer cell lines. We discovered that miR-22 gene consists of three exons spanning over 4.5 kb in the C17orf91 putative gene in chromosome 17. Forced expression of Twist increased miR-22 expression while knockdown of Twist lowered miR-22 expression in vitro. Consequently, forced expression of Twist and miR-22 lowered expression of ER in MCF-7 as seen at both RNA and protein levels. Mechanistically, both miR-22 binding sites in the ER 3’-UTR were important for miR-22 functionality and removal of either site was sufficient to abrogate ER downregulation. Finally, we observed that miR-22 expression was inversely proportional to ER expression and directly proportional to Twist expression in human breast cancer samples. Conclusion: These results suggest that Twist repression of ER is mediated by miR-22. The mechanism is by direct binding of Twist to E-boxes in the miR-22 promoter causing it to be upregulated. The dysregulated miR-22 binds to the 3’-UTR of ER leading to post-transcriptional downregulation and loss of ER. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 270. doi:10.1158/1538-7445.AM2011-270

Promoter Structure and Transcriptional Regulation of Human β-Galactoside α2, 3-Sialyltransferase Genes

Six human beta-galactoside alpha2,3-sialyltransferase genes, which are hST3Gal I-VI, have been cloned. Multiple genes encode enzymes with closely related catalytic specificities but different patterns of tissue expression. The multiple genes correspond to the control of various tissue specific regulators. Several studies have examined the transcriptional regulation of some human beta-galactoside alpha2,3-sialyltransferases genes. Multiple mRNA forms differing only in the 5'-untranslated regions have been identified in hST3Gal II, hST3Gal III, hST3Gal IV, hST3Gal V, and hST3Gal VI. These transcripts are produced by a combination of alternative splicing and promoter utilization, suggesting the transcriptional regulation of this gene depends on the use of alternative promoters, further suggesting that tissue-specific transcriptional regulation of these genes depends on the use of multiple genes and multiple promoters. The multiple regulatory pathways of these ubiquitous sialyltransferases may be differentially modulated in various cell types.

Alternative mRNA Splicing of SMRT Creates Functional Diversity by Generating Corepressor Isoforms with Different Affinities for Different Nuclear Receptors

Many eukaryotic transcription factors are bimodal in their regulatory properties and can both repress and activate expression of their target genes. These divergent transcriptional properties are conferred through recruitment of auxiliary proteins, denoted coactivators and corepressors. Repression plays a particularly critical role in the functions of the nuclear receptors, a large family of ligand-regulated transcription factors involved in metazoan development, differentiation, reproduction, and homeostasis. The SMRT corepressor interacts directly with nuclear receptors and serves, in turn, as a platform for the assembly of a larger corepressor complex. We report here that SMRT is expressed in cells by alternative mRNA splicing to yield two distinct variants or isoforms. We designate these isoforms SMRTalpha and SMRTtau and demonstrate that these isoforms have significantly different affinities for different nuclear receptors. These isoforms are evolutionarily conserved and are expressed in a tissue-specific manner. Our results suggest that differential mRNA splicing serves to customize corepressor function in different cells, allowing the transcriptional properties of nuclear receptors to be adapted to different contexts.

5종류의 인간유래 시알산전이효소 유전자들의 게놈구조 분석

인간유래 시알산전이효소 유전자들의 특이적 발현과 그들의 mRNA isoform의 생성에 대한 조절기구를 이해하기 위하여 5종류의 human 시알산전이효소 유전자(hST3Cal II, hST8Sia II, hST8Sia III, hSTS8Sia IV, hST8Sia V)들의 게놈구조를 분석하였다. hST3Gal II 유전자는 17 kb이상의 게놈상에 46 bp에서 1017 bp의 길이를 가진 exon이 6개로 이루어져 있고, hST8Sia III유전자는 10 kb이상의 게놈상에 125bp에서 2023bp의 길이를 가진 exon이 4개로 이루어져 있어 다른 human 시알산전이효소 유전자들보다 짧고 단순한 구조를 가지고 있었다. 반면에 다른 3종류의 유전자(hST8Sia II, hST8Sia IV, hST8Sia V)들은 70 kb이상의 게놈상에 5개이상의 exon으로 이루어져 있으며, 5종류 모두 exon-intron boundary는 GT-AG rule을 나타내고 있었다. 특히 모든 시알산전이효소에 고도로 보존되어 있는 sialylmotif L은 hST8Sia III유전자에서는 하나의 exon에 존재하는 반면에, 다른 시알산전이효소 유전자에서는 분리된 exon에 존재하여 exon의 구조적 다양성을 나타내고 있다. 또한, 본 연구에서는 5'-RACE와 cap site hunting법에 의해 hST3Gal II 유전자의 전사개시점을 결정하였다. Sialyltransferases cloned so far show the remarkable tissue-specific expression, which is correlated with the existence of cell type-specific sialylated sugar structure in glycoconjugates. In the previous studies, we found various mRNA isoforms of human sialyltransferases generated by alternative splicing and alternative promoter utilization. To understand the regulatory mechanisms for specific expression of human sialyltransferase genes and for production of their mRNA isoforms, in this study, we have isolated and characterized five kinds of human sialyltransferase genes: hST3Gal II, hST8Sia II, hST8Sia III, hST8Sia IV, and hST8Sia V. The hST3Gal II gene is composed of six exons, which span over 17kb, with exons ranging in size from 46 to over 1017 bp. The hST8Sia III gene comprises over 10 kb, and consists of only four exons, which is much smaller and simpler than other human sialyltransferase genes. In contrast, three genes (hST8Sia II, hST8Sia IV and hST8Sia V) span more than 70 kb, and comprise five or more exons. All exon-intron boundaries follow the GT-AG rule. In particular, the sialylmotif L, which is a highly conserved region in all cloned sialyltransferases, was found in one exon of hST8Sia III, whereas this motif is encoded by discrete exons in the other human sialyltransferases. Exon structures of these sialyltransferase genes show the structural diversity, as found in other human sialyltransferase genes reported so far. We determined the transcription start site of hST3Gal II gene by the 5'-RACE and cap site hunting experiments.

Genomic structure, expression, and transcriptional regulation of human Gal [formula omitted]1,3 GalNAc [formula omitted]2,3-sialyltransferase gene

In this report, we describe transcriptional regulation of the human Gal β1,3 GalNAc α2,3-sialyltransferase II (hST3Gal II) gene. The results of 5 ′-RACE showed that the forms of two mRNAs differed only in the 5 ′-untranslated region (Types 1 and 2). According to analysis of the genomic structure, the transcriptional regulation of Type 1 and Type 2 mRNA isoforms depended on the p1 and p2 promoters, respectively. Both the mRNA isoforms were detected in various human tissues except colon, skeletal muscle, and peripheral blood leukocytes by RT-PCR analysis. In colon tissue, the Type 2 mRNA was detected, however, Type 1 mRNA was not detected. To elucidate the molecular basis of hST3Gal II gene expression, we isolated and characterized the function of the genomic region of hST3Gal II containing the p1 and p2 promoters. The activity of p2 promoter is much higher than that of the p1 promoter in the colon adenocarcinoma cell line, COLO205. These results suggest that the hST3Gal II gene is expressed specifically by alternative promoter utilization and is regulated in a tissue-restricted fashion at the level of transcription.

Immunocytochemical Localization of Anion Exchanger-2 (AE-2) in the Dorsal Root Ganglion of Adult Rats

Nearly a century ago, Otto Warburg made the seminal observation that tumors metabolize glucose more rapidly than normal tissue and do so even in the presence of ox-ygen [1,2]. Since this observation, numerous studies have addressed how cancer cells enhance glucose uptake and the mechanisms by which this additional glucose is utilized [3,4]. Several lines of evidence suggest that can-cer cells reprogram their metabolic networks through coordinated regulation of enzyme expression and activi-ty to enhance anabolic processes. One process that influ-ences expression of metabolic enzymes important in cancer is alternative splicing. Among the alternatively spliced genes expressed in cancer cells is the embryonic isoform of pyruvate kinase, PKM2 [5]. This observation was pioneered by Erich Eigenbrodt and is now consid-ered as a major node in the regulation of cancer metabo-lism [6,7]. In the following commentary, we describe the mechanisms by which the activity of PKM2 is controlled in tumors to facilitate cell growth, work that has stemmed from Eigenbrodt’s efforts exploring the onco-genic role of this glycolytic isozyme. Pyruvate kinase (PK) is a glycolytic enzyme that converts phosphoenolpyruvate (PEP) and ADP into pyruvate and ATP. This reaction is both a rate-limiting step and the final step in glycolysis [6]. In mammals, there are two genes that encode four PK isozymes [8]. Alternative pro-moter utilization of the

Gene Structure and Transcriptional Regulation of Human Gal β1,4(3) GlcNAc α2,3-Sialyltransferase VI (hST3Gal VI) Gene in Prostate Cancer Cell Line

We describe transcriptional regulation of the human Gal β1,4(3) GlcNAc α2,3-sialyltransferase VI (hST3Gal VI) gene. The 5′-RACE results indicated that two mRNA forms differ only in the 5′-untranslated region (types 1 and 2). The genomic structure shows that the transcriptional regulation of type 1 and type 2 mRNA depends on the P1 and P2 promoters, respectively. Northern blots of RNA derived from various human tissues showed that the expression level of type 2 mRNA is higher than that of type 1 in the prostate. To elucidate the molecular basis of hST3Gal VI gene expression, we isolated and functionally characterized the genomic region containing the P1 and P2 promoters of hST3Gal VI. The activity of the P2 promoter is much higher than that of the P1 promoter in the prostate adenocarcinoma cell line PC-3. The results suggested that the hST3Gal VI gene is expressed specifically by alternative promoter utilization and is regulated in a tissue-restricted fashion at the level of transcription.

Genomic structure of human GM3 synthase gene (hST3Gal V) and identification of mRNA isoforms in the 5′-untranslated region

GM3 synthase, which transfers CMP-NeuAc with an α2,3-linkage to a galactose residue of lactosylceramide, plays a key role in the biosynthesis of all complex gangliosides. In this study, cDNA and genomic clones encoding human GM3 synthase (hST3Gal V) were isolated, and the structural organization of the gene was determined. The hST3Gal V cDNA was identical in the coding region with cDNA that has been cloned previously from the HL-60 cells but different in the 5′-untranslated region (UTR). The hST3Gal V gene consisted of nine exons, which span approximately 44 kb, with exons ranging in size from 112 to 1242 bp. The coding region was located in exons 4–9, and all exon–intron boundaries except the acceptor site of intron 1 followed the GT-AG rule. The expression of this gene was highly restricted in both human fetal and adult tissues. By comparison of the nucleotide sequences of the genomic DNA with cDNA sequences including 5′-RACE products, we identified four isoforms (types 1–4) of the hST3Gal V mRNA that differ only in the 5′-UTR. Structural analysis of these isoforms suggests that mRNA isoforms of hST3Gal V are produced by a combination of alternative splicing and alternative promoter utilization.

Tissue-specific and Ubiquitous Promoters Direct the Expression of Alternatively Spliced Transcripts from the Calcitonin Receptor Gene

The gene encoding the murine calcitonin receptor (mCTR) was isolated, and the exon/intron structure was determined. Analysis of transcripts revealed novel cDNA sequences, new alternative exon splicing in the 5'-untranslated region, and three putative promoters (P1, P2, and P3). The longest transcription unit is greater than 67 kilobase pairs, and the location of introns within the coding region of the mCTR gene (exons E3-E14) are identical to those of the porcine and human CTR genes. We have identified novel cDNA sequences that form three new exons as well as others that add 512 base pairs to the 5' side of the previously published cDNA, thereby extending exon E1 to 682 base pairs. Two of these novel exons are upstream of exon E2 and form a tripartite exon E2 (E2a, E2b, and E2c) in which E2a is utilized by promoter P2 with variable splicing of E2b. The third new exon (E3b') lies between E3a and E3b and is utilized by promoter P3. Analysis of mCTR mRNAs has revealed that the three alternative promoters give rise to at least seven mCTR isoforms in the 5' region of the gene and generate 5'-untranslated regions of very different lengths. Analysis by reverse transcription-polymerase chain reaction shows that promoters P1 and P2 are utilized in osteoclasts, brain, and kidney, whereas promoter P3 appears to be osteoclast-specific. Using transiently transfected reporter constructs, promoter P2 has activity in both a murine kidney cell line (MDCT209) and a chicken osteoclast-like cell line (HD-11EM), whereas promoter P3 is active only in the osteoclast-like cell line. These transfection data confirm the osteoclast specificity of promoter P3 and provide the first evidence that the CTR gene is regulated in a tissue-specific manner by alternative promoter utilization.

Mammalian aromatases

Aromatase is the enzyme complex that catalyses the synthesis of oestrogens from androgens, and therefore it has unique potential to influence the physiological balance between the sex steroid hormones. Both aromatase cytochrome P450 (P450arom) and NADPH-cytochrome P450 reductase (reductase), the two essential components of the enzyme complex, are highly conserved among mammals and vertebrates. Aromatase expression occurs in the gonads and brain, and is essential for reproductive development and fertility. Of interest are the complex mechanisms involving alternative promoter utilization that have evolved to control tissue-specific expression in these tissues. In addition, in a number of species, including humans, expression of aromatase has a broader tissue distribution, including placenta, adipose and bone. The relevance of oestrogen synthesis and possibly androgen metabolism in these peripheral sites of expression is now becoming clear from studies in P450arom knockout (ArKO) mice and from genetic defects recognized recently in both men and women. Important species differences in the physiological roles of aromatase expression are also likely to emerge, despite the highly conserved nature of the enzyme system. The identification of functionally distinct, tissue-specific isozymes of P450arom in at least one mammal, pigs, and several species of fish indicates that there are additional subtle, but physiologically significant, species-specific roles for aromatase. Comparative studies of mammalian and other vertebrate aromatases will expand understanding of the role played by this ancient enzyme system in the evolution of reproduction and the adaptive influence of oestrogen synthesis on general health and well being.

The human prolactin receptor gene structure and alternative promoter utilization: the generic promoter hPIII and a novel human promoter hP(N).

The 5'-untranslated region of the human prolactin receptor (hPRLR) gene contains two alternative first exons, hE1(3), the human counterpart of the rat and mouse E1(3) and a novel human type of alternative first exon termed hE1N, also a common non-coding exon 2 and a third exon containing the translation initiation codon. hE1(3) was localized approximately 800 bp 5' from the hE1N in the genome. The two distinct first exons hE1(3) and hE1N are expressed in human breast tissue, breast cancer cells, gonads and liver. Overall, the transcript containing hE1(3) is prevalent in most tissues. The coding region of the gene comprises eight exons (exon 3-10), in which exon 10 encodes most of the intracellular domain. hE1(3) and hE1N are transcribed from alternative promoters hPIII and hP(N), respectively. The hPIII, containing identical Sp1 and C/EBP elements as in the rodent promoters, shares 81% similarity in the region -480/-106 to both the rat and mouse. The novel promoter hP(N) contains putative binding sites for ETS-family proteins and a half-site for nuclear receptors. Therefore, both promoters likely utilize distinct mechanisms in controlling the hPRLR gene transcription. The different promoter utilization of the hPRLR gene in diverse tissues may confer differential prolactin response through activation of different promoters.

The Human Prolactin Receptor Gene Structure and Alternative Promoter Utilization: The Generic Promoter hPII and a Novel Human Promoter hPN

The 5′-untranslated region of the human prolactin receptor (hPRLR) gene contains two alternative first exons, hE13, the human counterpart of the rat and mouse E13, and a novel human type of alternative first exon termed hE1N, also a common non-coding exon 2 and a third exon containing the translation initiation codon. hE13 was localized ∼800 bp 5′ from the hE1N in the genome. The two distinct first exons hE13 and hE1N are expressed in human breast tissue, breast cancer cells, gonads and liver. Overall, the transcript containing hE13 is prevalent in most tissues. The coding region of the gene comprises eight exons (exons 3–10), in which exon 10 encodes most of the intracellular domain. hE13 and hE1N are transcribed from alternative promoters hPIII and hPN, respectively. The hPIII, containing identical Sp1 and C/EBP elements as in the rodent promoters, shares 81% similarity in the region −480/−106 to both the rat and mouse. The novel promoter hPN contains putative binding sites for ETS-family proteins and a half-site for nuclear receptors. Therefore, both promoters likely utilize distinct mechanisms in controlling the hPRLR gene transcription. The different promoter utilization of the hPRLR gene in diverse tissues may confer differential prolactin response through activation of different promoters.

Sequence of interleukin-2 isolated from human placental poly A+ RNA: possible role in maintenance of fetal allograft.

There are several cell types within the placenta that produce cytokines which can contribute to the regulatory mechanisms that ensure normal pregnancy. The immunological milieu at the maternofetal interface is considered to be crucial for survival of the fetus. Interleukin-2 (IL-2) is expressed by the syncytiotrophoblast, the cell layer between the mother and the fetus. IL-2 appears to be a key factor in maintenance of pregnancy. Therefore, it was important to determine the sequence of human placental interleukin-2. Direct sequencing of human placental IL-2 cDNA was determined for the coding region. Subclone sequencing was carried out for the 5'- and 3'-untranslated regions (5'-UTR and 3'-UTR). The 5'-UTR for human placental IL-2 cDNA is 294 bp, which is 247 nucleotides longer than that reported for cDNA IL-2 derived from T cells. The sequence of the coding region is identical to that reported for T cell IL-2, while sequence analysis of the polymerase chain reaction (PCR) product showed that the cDNA from the 3' end was the same as that reported for cDNA from T cells. Human placental IL-2 cDNA is 1,028 base pairs (excluding the poly A tail), which is 247 bp longer at the 5' end than that reported for IL-2 T cell cDNA. Therefore, the extended 5'-UTR of the placental IL-2 cDNA may be a consequence of alternative promoter utilization in the placenta.

Genomic Organization and Chromosomal Mapping of the Galβ1,3GalNAc/Galβ1,4GlcNAc α2,3-Sialyltransferase

In this report we describe the chromosome mapping and genomic organization of the human Gal beta 1,3GalNAc/Gal beta 1,4GlcNAc alpha 2,3-sialyltransferase gene. The gene is localized to human chromosome 11(q23-q24) by in situ hybridization of metaphase chromosomes. It spans more than 25 kilobases of human genomic DNA and is distributed over 14 exons that range in size from 61 to 679 base pairs. Previous characterization of cDNAs encoding the Gal beta 1,3GalNAc/Gal beta 1,4GlcNAc alpha 2,3-sialyltransferase revealed that the gene produces at least three transcripts in human placenta, which code for identical protein sequences except at the 5' ends (Kitagawa, H., and Paulson, J. C. (1994a) J. Biol. Chem. 269, 1394-1401). Repeated screening for clones that contain the 5' end of the cDNA has identified two additional distinct mRNAs that are expressed in human placenta. Comparison of the genomic DNA sequence with that of the five different mRNAs indicates that these transcripts are produced by a combination of alternative splicing and alternative promoter utilization. Northern analysis indicated that one of them is specifically expressed in placenta, testis, and ovary, indicating that its expression is independently regulated from the others.

Estrogen target tissue determines alternative promoter utilization of the human estrogen receptor gene in osteoblasts and tumor cell lines

Estrogen target tissue determines alternative promoter utilization of the human estrogen receptor gene in osteoblasts and tumor cell lines

Estrogen target tissue determines alternative promoter utilization of the human estrogen receptor gene in osteoblasts and tumor cell lines.

The estrogen receptor (ER) mediates the effects of its cognate ligand on important cellular processes such as development of female secondary sexual characteristics, establishment and maintenance of pregnancy, progression of breast cancer, and maintenance of bone mass. We have previously demonstrated that the human ER (hER) gene is transcribed from two promoters, suggesting that tissue- and cell-specific expression patterns of this gene may, at least in part, be regulated by differential promoter usage. Here we show, by using a reverse transcriptase coupled polymerase chain reaction assay, that transcripts initiated from both hER gene promoters were expressed in breast and uterus. In contrast, only transcripts originating from the distal promoter could be detected in human primary osteoblasts. Furthermore, determination of the expression levels of the two hER transcripts by quantitative polymerase chain reaction demonstrated an almost 30-fold increase of the transcripts originating from the proximal promoter in breast cancer cell lines over that detected in normal breast tissue. Taken together, our results demonstrate a previously unrecognized mechanism for regulation of hER gene expression by tissue-specific differential promoter utilization. In addition, our results suggest that estrogen-dependent cell transformation may be accompanied by a change in the relative expression levels of the two hER transcripts.

The PITSLRE protein kinase family.

A family of p34Cdc2 related protein kinases, the PITSLRE kinases, is generated by alternative splicing and promoter utilization from three duplicated and tandemly linked genes on human chromosome 1p36.3, which is frequently deleted during the late stages of tumorigenesis. PITSLRE mRNA, protein, and enzyme activity are induced during Fas receptor- and glucocorticoid-mediated apoptosis of human T cells. Several PITSLRE isoforms are specific targets of proteolysis during apoptosis, generating an enzymatically active 50 kDa isoform. Inhibition of this protease activity blocks PITSLRE processing and enzyme activation, as well as apoptosis. Thus, PITSLRE kinases may be integral downstream components of apoptotic signal transduction pathway(s). Furthermore, PITSLRE genes, and their products, are physically altered in human neuroblastoma tumors, suggesting that they may be tumor suppressors.