Genome-wide association studies (GWASs) have linked genes to various pathological traits. However... more Genome-wide association studies (GWASs) have linked genes to various pathological traits. However, the potential contribution of regulatory noncoding RNAs, such as microRNAs (miRNAs), to a genetic predisposition to pathological conditions has remained unclear. We leveraged GWAS meta-analysis data from >188,000 individuals to identify 69 miRNAs in physical proximity to single-nucleotide polymorphisms (SNPs) associated with abnormal levels of circulating lipids. Several of these miRNAs (miR-128-1, miR-148a, miR-130b, and miR-301b) control the expression of key proteins involved in cholesterol-lipoprotein trafficking, such as the low-density lipoprotein (LDL) receptor (LDLR) and the ATP-binding cassette A1 (ABCA1) cholesterol transporter. Consistent with human liver expression data and genetic links to abnormal blood lipid levels, overexpression and antisense targeting of miR-128-1 or miR-148a in high-fat diet-fed C57BL/6J and Apoe-null mice resulted in altered hepatic expression of...
Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the ... more Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targeting miRNAs. This indirect but highly specific mechanism, by which a chromatin regulator that mediates transcriptional activating marks can lead to the downregulation of a critical effector gene, is shared with multiple genes in the p53 pathway. Through such miRNA-dependent effects, SETD1A regulates cell cycle progression in vitro and modulates tumorigenesis in mouse xenograft models. Together, these observations help explain the remarkably specific genetic consequences associated with alterations in generic chromatin modulators in cancer.
Our previous study identified two alternate non-coding upstream exons (A and B) in the human redu... more Our previous study identified two alternate non-coding upstream exons (A and B) in the human reduced folate carrier (hRFC) gene, each controlled by a separate promoter. Each minimal promoter was regulated by unique cis -elements and transcription factors, including stimulating protein (Sp) 1 and Sp3 and the basic leucine zipper family of proteins, suggesting opportunities for cell- and tissue-specific regulation. Studies were performed to explore the expression patterns of hRFC in human tissues and cell lines. Levels of hRFC transcripts were measured on a multi-tissue mRNA array from 76 human tissues and tumour cell lines and on a multi-tissue Northern blot of representative tissues, each probed with full-length hRFC cDNA. hRFC transcripts were ubiquitously expressed, with the highest level in placenta and the lowest level in skeletal muscle. By rapid amplification of cDNA 5'-ends assay from nine tissues and two cell lines, hRFC transcripts containing both A and B 5'-untrans...
Epithelial-mesenchymal transition (EMT) is thought to contribute to cancer metastasis, but its un... more Epithelial-mesenchymal transition (EMT) is thought to contribute to cancer metastasis, but its underlying mechanisms are not well understood. To define early steps in this cellular transformation, we analyzed human mammary epithelial cells with tightly regulated expression of Snail-1, a master regulator of EMT. After Snail-1 induction, epithelial markers were repressed within 6 hr, and mesenchymal genes were induced at 24 hr. Snail-1 binding to its target promoters was transient (6-48 hr) despite continued protein expression, and it was followed by both transient and long-lasting chromatin changes. Pharmacological inhibition of selected histone acetylation and demethylation pathways suppressed the induction as well as the maintenance of Snail-1-mediated EMT. Thus, EMT involves an epigenetic switch that may be prevented or reversed with the use of small-molecule inhibitors of chromatin modifiers.
The flavin-containing monooxygenases (FMOs) are a family of xenobiotic-metabolizing enzymes that ... more The flavin-containing monooxygenases (FMOs) are a family of xenobiotic-metabolizing enzymes that are expressed in a species- and tissue-specific manner. FMO2 expression has been observed in pulmonary tissue from several species, but not human. Two human FMO2 point mutations have been reported: a cytosine to thymidine transition at position 1414 resulting in a premature stop codon and a thymidine insertion at position 1589 resulting in a frameshift. To define the frequency of these sequence variations and explore their significance, unrelated African-American, Caucasian, and Korean individuals were genotyped. In the African-American population tested (n = 180), the 1414C allele occurred at a 13% frequency; however, all of the tested Caucasians (n = 52) and Koreans (n = 100) were homozygous for the 1414T allele. The T1589 allele occurred at frequencies of 6.9 and 13.0% in African-Americans (n = 175) and Caucasians (n = 23), respectively, and appears to segregate with the 1414T allele. Thus, it would have no further impact on FMO2 activity. Western blot analysis of pulmonary microsomes failed to detect immunoreactive protein in 1414T homozygotes. A heterozygotic individual did exhibit a single band of the expected size, but no detectable FMO activity in the corresponding lung microsomes. Sequence analysis, however, was consistent with the 1414C allele encoding an active FMO2 enzyme. FMO2 mRNA expression was observed in most individuals, but failed to correlate with genotype or protein expression. In summary, functional FMO2 is expressed in only a small percentage of the overall population. However, in certain ethnic groups, active pulmonary FMO2 enzyme will be present in a significant number of individuals.
Proceedings of the National Academy of Sciences, 2009
CtBP (C-terminal binding protein) is an evolutionarily conserved NAD(H)-dependent transcriptional... more CtBP (C-terminal binding protein) is an evolutionarily conserved NAD(H)-dependent transcriptional corepressor, whose activity has been shown to be regulated by the NAD/NADH ratio. Although recent studies have provided significant new insights into mechanisms by which CtBP regulates transcription, the biological function of CtBP remains incompletely understood. Here, we report that genetic inactivation of the Caenorhabditis elegans homolog, ctbp-1, results in life span extension, which is suppressed by reintroduction of the ctbp-1 genomic DNA encoding wild-type but not NAD(H)-binding defective CTBP-1 protein. We show that CTBP-1 possibly modulates aging through the insulin/IGF-1 signaling pathway, dependent on the forkhead transcription factor DAF-16, but independent of the NAD-dependent histone deacetylase SIR-2.1. Genome-wide microarray analysis identifies >200 potential CTBP-1 target genes. Importantly, RNAi inhibition of a putative triacylglycerol lipase gene lips-7(C09E8.2) but not another lipase suppresses the life span extension phenotype. Consistently, metabolic analysis shows that the triacylglycerol level is reduced in the ctbp-1 deletion mutant, which is restored to the wild-type level by RNAi inhibition of lips-7. Taken together, our data suggest that CTBP-1 controls life span probably through the regulation of lipid metabolism.
... S. Jacobshagen, JR Whetstine, and JM Boling 594 ... Such an expression pattern, however, seem... more ... S. Jacobshagen, JR Whetstine, and JM Boling 594 ... Such an expression pattern, however, seems unlikely because both proteins are needed for the assembly of a functional ribosome (Finley et al., 1989[9]). We also cannot eliminate the possibility that the mRNA half-lives are too ...
The recent discovery of a large number of histone demethylases suggests a central role for these ... more The recent discovery of a large number of histone demethylases suggests a central role for these enzymes in regulating histone methylation dynamics. Histone H3K27 trimethylation (H3K27me3) has been linked to polycomb-group-protein-mediated suppression of Hox genes and animal body patterning, X-chromosome inactivation and possibly maintenance of embryonic stem cell (ESC) identity. An imbalance of H3K27 methylation owing to overexpression of the methylase EZH2 has been implicated in metastatic prostate and aggressive breast cancers. Here we show that the JmjC-domain-containing related proteins UTX and JMJD3 catalyse demethylation of H3K27me3/2. UTX is enriched around the transcription start sites of many HOX genes in primary human fibroblasts, in which HOX genes are differentially expressed, but is selectively excluded from the HOX loci in ESCs, in which HOX genes are largely silent. Consistently, RNA interference inhibition of UTX led to increased H3K27me3 levels at some HOX gene promoters. Importantly, morpholino oligonucleotide inhibition of a zebrafish UTX homologue resulted in mis-regulation of hox genes and a striking posterior developmental defect, which was partially rescued by wild-type, but not by catalytically inactive, human UTX. Taken together, these findings identify a small family of H3K27 demethylases with important, evolutionarily conserved roles in H3K27 methylation regulation and in animal anterior-posterior development.
In the current issue of Molecular Cell, Herranz et al. (2012) demonstrate that LOXL2 deaminates t... more In the current issue of Molecular Cell, Herranz et al. (2012) demonstrate that LOXL2 deaminates trimethylated histone 3 lysine 4 (H3K4me3), which uncovers a new chromatin modification and a new enzymatic mechanism with the potential to regulate additional lysine residues.
Recently, our laboratory reported an intricate regulation of the human reduced folate carrier (hR... more Recently, our laboratory reported an intricate regulation of the human reduced folate carrier (hRFC) gene, involving multiple promoters and noncoding exons. We localized promoter activity to a 452-bp GC-rich region upstream of noncoding exon A, including a 47-bp basal promoter with a CRE/AP-1-like consensus element that bound the bZip family of DNA-binding proteins (e.g. CREB-1 and c-Jun). We now report that three nearly identical tandem repeats (49-61 bp) in the hRFC-A upstream region are involved in regulating promoter activity. By in vitro binding assays, multiple transcription factors (e.g. AP2 and Sp1/Sp3) bound this region. When AP2 was cotransfected with the hRFC-A reporter construct into HT1080 cells, promoter activity increased 3-fold. In Drosophila SL2 cells, Sp1 transactivated promoter A and showed synergism with CREB-1. However, c-Jun was antagonistic to the effects of Sp1. A sequence variant in the hRFC-A repeated region was identified, involving an exact duplication of a 61-bp sequence. This variant had an allelic frequency of 78% in 72 genomic DNAs and resulted in a 63% increase in promoter activity. These results identify important regions in the hRFC-A promoter and critical roles for AP2 and Sp1, in combination with the bZip transcription factors. Moreover, they document a functionally novel polymorphism that increases promoter activity and may contribute to interpatient variations in hRFC expression and effects on tissue folate homeostasis and antitumor response to antifolates.
Our laboratory previously identified two functional promoters (designated A and B) for the human ... more Our laboratory previously identified two functional promoters (designated A and B) for the human reduced folate carrier (hRFC) gene that result in hRFC transcripts with differing 5'-untranslated regions. By transiently transfecting HT1080 and HepG2 cells with a series of 5' and 3' deletions in the hRFC-B and -A promoters, the minimal promoters were localized within 46 and 47 base pairs, respectively. Gel mobility shift assays with the hRFC-B basal promoter region revealed specific DNA-protein complexes involving a highly conserved GC-box and Sp1 or Sp3. In Drosophila SL2 cells, both Sp1 and the long Sp3 isoform potently transactivated the hRFC-B basal promoter; however, the short Sp3 isoforms were transcriptionally inert and resulted in a potent inhibition of Sp1 transactivation. For the hRFC-A basal promoter, a CRE/AP-1-like element was bound by the bZip superfamily of DNA-binding proteins. Cell-specific DNA-protein complexes were identified for hRFC-A (CREB-1 and c-Jun in HT1080 cells; CREB-1 and ATF-1 in HepG2 cells). When the GC-box and CRE/AP-1-like elements were mutated, a 60--90% decrease in promoter activity was observed in both cell lines. These results identify the critical regulatory regions for the hRFC basal promoters and stress the functional importance of the Sp and bZip families of transcription factors in regulating hRFC expression.
Genome-wide association studies (GWASs) have linked genes to various pathological traits. However... more Genome-wide association studies (GWASs) have linked genes to various pathological traits. However, the potential contribution of regulatory noncoding RNAs, such as microRNAs (miRNAs), to a genetic predisposition to pathological conditions has remained unclear. We leveraged GWAS meta-analysis data from >188,000 individuals to identify 69 miRNAs in physical proximity to single-nucleotide polymorphisms (SNPs) associated with abnormal levels of circulating lipids. Several of these miRNAs (miR-128-1, miR-148a, miR-130b, and miR-301b) control the expression of key proteins involved in cholesterol-lipoprotein trafficking, such as the low-density lipoprotein (LDL) receptor (LDLR) and the ATP-binding cassette A1 (ABCA1) cholesterol transporter. Consistent with human liver expression data and genetic links to abnormal blood lipid levels, overexpression and antisense targeting of miR-128-1 or miR-148a in high-fat diet-fed C57BL/6J and Apoe-null mice resulted in altered hepatic expression of...
Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the ... more Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targeting miRNAs. This indirect but highly specific mechanism, by which a chromatin regulator that mediates transcriptional activating marks can lead to the downregulation of a critical effector gene, is shared with multiple genes in the p53 pathway. Through such miRNA-dependent effects, SETD1A regulates cell cycle progression in vitro and modulates tumorigenesis in mouse xenograft models. Together, these observations help explain the remarkably specific genetic consequences associated with alterations in generic chromatin modulators in cancer.
Our previous study identified two alternate non-coding upstream exons (A and B) in the human redu... more Our previous study identified two alternate non-coding upstream exons (A and B) in the human reduced folate carrier (hRFC) gene, each controlled by a separate promoter. Each minimal promoter was regulated by unique cis -elements and transcription factors, including stimulating protein (Sp) 1 and Sp3 and the basic leucine zipper family of proteins, suggesting opportunities for cell- and tissue-specific regulation. Studies were performed to explore the expression patterns of hRFC in human tissues and cell lines. Levels of hRFC transcripts were measured on a multi-tissue mRNA array from 76 human tissues and tumour cell lines and on a multi-tissue Northern blot of representative tissues, each probed with full-length hRFC cDNA. hRFC transcripts were ubiquitously expressed, with the highest level in placenta and the lowest level in skeletal muscle. By rapid amplification of cDNA 5'-ends assay from nine tissues and two cell lines, hRFC transcripts containing both A and B 5'-untrans...
Epithelial-mesenchymal transition (EMT) is thought to contribute to cancer metastasis, but its un... more Epithelial-mesenchymal transition (EMT) is thought to contribute to cancer metastasis, but its underlying mechanisms are not well understood. To define early steps in this cellular transformation, we analyzed human mammary epithelial cells with tightly regulated expression of Snail-1, a master regulator of EMT. After Snail-1 induction, epithelial markers were repressed within 6 hr, and mesenchymal genes were induced at 24 hr. Snail-1 binding to its target promoters was transient (6-48 hr) despite continued protein expression, and it was followed by both transient and long-lasting chromatin changes. Pharmacological inhibition of selected histone acetylation and demethylation pathways suppressed the induction as well as the maintenance of Snail-1-mediated EMT. Thus, EMT involves an epigenetic switch that may be prevented or reversed with the use of small-molecule inhibitors of chromatin modifiers.
The flavin-containing monooxygenases (FMOs) are a family of xenobiotic-metabolizing enzymes that ... more The flavin-containing monooxygenases (FMOs) are a family of xenobiotic-metabolizing enzymes that are expressed in a species- and tissue-specific manner. FMO2 expression has been observed in pulmonary tissue from several species, but not human. Two human FMO2 point mutations have been reported: a cytosine to thymidine transition at position 1414 resulting in a premature stop codon and a thymidine insertion at position 1589 resulting in a frameshift. To define the frequency of these sequence variations and explore their significance, unrelated African-American, Caucasian, and Korean individuals were genotyped. In the African-American population tested (n = 180), the 1414C allele occurred at a 13% frequency; however, all of the tested Caucasians (n = 52) and Koreans (n = 100) were homozygous for the 1414T allele. The T1589 allele occurred at frequencies of 6.9 and 13.0% in African-Americans (n = 175) and Caucasians (n = 23), respectively, and appears to segregate with the 1414T allele. Thus, it would have no further impact on FMO2 activity. Western blot analysis of pulmonary microsomes failed to detect immunoreactive protein in 1414T homozygotes. A heterozygotic individual did exhibit a single band of the expected size, but no detectable FMO activity in the corresponding lung microsomes. Sequence analysis, however, was consistent with the 1414C allele encoding an active FMO2 enzyme. FMO2 mRNA expression was observed in most individuals, but failed to correlate with genotype or protein expression. In summary, functional FMO2 is expressed in only a small percentage of the overall population. However, in certain ethnic groups, active pulmonary FMO2 enzyme will be present in a significant number of individuals.
Proceedings of the National Academy of Sciences, 2009
CtBP (C-terminal binding protein) is an evolutionarily conserved NAD(H)-dependent transcriptional... more CtBP (C-terminal binding protein) is an evolutionarily conserved NAD(H)-dependent transcriptional corepressor, whose activity has been shown to be regulated by the NAD/NADH ratio. Although recent studies have provided significant new insights into mechanisms by which CtBP regulates transcription, the biological function of CtBP remains incompletely understood. Here, we report that genetic inactivation of the Caenorhabditis elegans homolog, ctbp-1, results in life span extension, which is suppressed by reintroduction of the ctbp-1 genomic DNA encoding wild-type but not NAD(H)-binding defective CTBP-1 protein. We show that CTBP-1 possibly modulates aging through the insulin/IGF-1 signaling pathway, dependent on the forkhead transcription factor DAF-16, but independent of the NAD-dependent histone deacetylase SIR-2.1. Genome-wide microarray analysis identifies >200 potential CTBP-1 target genes. Importantly, RNAi inhibition of a putative triacylglycerol lipase gene lips-7(C09E8.2) but not another lipase suppresses the life span extension phenotype. Consistently, metabolic analysis shows that the triacylglycerol level is reduced in the ctbp-1 deletion mutant, which is restored to the wild-type level by RNAi inhibition of lips-7. Taken together, our data suggest that CTBP-1 controls life span probably through the regulation of lipid metabolism.
... S. Jacobshagen, JR Whetstine, and JM Boling 594 ... Such an expression pattern, however, seem... more ... S. Jacobshagen, JR Whetstine, and JM Boling 594 ... Such an expression pattern, however, seems unlikely because both proteins are needed for the assembly of a functional ribosome (Finley et al., 1989[9]). We also cannot eliminate the possibility that the mRNA half-lives are too ...
The recent discovery of a large number of histone demethylases suggests a central role for these ... more The recent discovery of a large number of histone demethylases suggests a central role for these enzymes in regulating histone methylation dynamics. Histone H3K27 trimethylation (H3K27me3) has been linked to polycomb-group-protein-mediated suppression of Hox genes and animal body patterning, X-chromosome inactivation and possibly maintenance of embryonic stem cell (ESC) identity. An imbalance of H3K27 methylation owing to overexpression of the methylase EZH2 has been implicated in metastatic prostate and aggressive breast cancers. Here we show that the JmjC-domain-containing related proteins UTX and JMJD3 catalyse demethylation of H3K27me3/2. UTX is enriched around the transcription start sites of many HOX genes in primary human fibroblasts, in which HOX genes are differentially expressed, but is selectively excluded from the HOX loci in ESCs, in which HOX genes are largely silent. Consistently, RNA interference inhibition of UTX led to increased H3K27me3 levels at some HOX gene promoters. Importantly, morpholino oligonucleotide inhibition of a zebrafish UTX homologue resulted in mis-regulation of hox genes and a striking posterior developmental defect, which was partially rescued by wild-type, but not by catalytically inactive, human UTX. Taken together, these findings identify a small family of H3K27 demethylases with important, evolutionarily conserved roles in H3K27 methylation regulation and in animal anterior-posterior development.
In the current issue of Molecular Cell, Herranz et al. (2012) demonstrate that LOXL2 deaminates t... more In the current issue of Molecular Cell, Herranz et al. (2012) demonstrate that LOXL2 deaminates trimethylated histone 3 lysine 4 (H3K4me3), which uncovers a new chromatin modification and a new enzymatic mechanism with the potential to regulate additional lysine residues.
Recently, our laboratory reported an intricate regulation of the human reduced folate carrier (hR... more Recently, our laboratory reported an intricate regulation of the human reduced folate carrier (hRFC) gene, involving multiple promoters and noncoding exons. We localized promoter activity to a 452-bp GC-rich region upstream of noncoding exon A, including a 47-bp basal promoter with a CRE/AP-1-like consensus element that bound the bZip family of DNA-binding proteins (e.g. CREB-1 and c-Jun). We now report that three nearly identical tandem repeats (49-61 bp) in the hRFC-A upstream region are involved in regulating promoter activity. By in vitro binding assays, multiple transcription factors (e.g. AP2 and Sp1/Sp3) bound this region. When AP2 was cotransfected with the hRFC-A reporter construct into HT1080 cells, promoter activity increased 3-fold. In Drosophila SL2 cells, Sp1 transactivated promoter A and showed synergism with CREB-1. However, c-Jun was antagonistic to the effects of Sp1. A sequence variant in the hRFC-A repeated region was identified, involving an exact duplication of a 61-bp sequence. This variant had an allelic frequency of 78% in 72 genomic DNAs and resulted in a 63% increase in promoter activity. These results identify important regions in the hRFC-A promoter and critical roles for AP2 and Sp1, in combination with the bZip transcription factors. Moreover, they document a functionally novel polymorphism that increases promoter activity and may contribute to interpatient variations in hRFC expression and effects on tissue folate homeostasis and antitumor response to antifolates.
Our laboratory previously identified two functional promoters (designated A and B) for the human ... more Our laboratory previously identified two functional promoters (designated A and B) for the human reduced folate carrier (hRFC) gene that result in hRFC transcripts with differing 5'-untranslated regions. By transiently transfecting HT1080 and HepG2 cells with a series of 5' and 3' deletions in the hRFC-B and -A promoters, the minimal promoters were localized within 46 and 47 base pairs, respectively. Gel mobility shift assays with the hRFC-B basal promoter region revealed specific DNA-protein complexes involving a highly conserved GC-box and Sp1 or Sp3. In Drosophila SL2 cells, both Sp1 and the long Sp3 isoform potently transactivated the hRFC-B basal promoter; however, the short Sp3 isoforms were transcriptionally inert and resulted in a potent inhibition of Sp1 transactivation. For the hRFC-A basal promoter, a CRE/AP-1-like element was bound by the bZip superfamily of DNA-binding proteins. Cell-specific DNA-protein complexes were identified for hRFC-A (CREB-1 and c-Jun in HT1080 cells; CREB-1 and ATF-1 in HepG2 cells). When the GC-box and CRE/AP-1-like elements were mutated, a 60--90% decrease in promoter activity was observed in both cell lines. These results identify the critical regulatory regions for the hRFC basal promoters and stress the functional importance of the Sp and bZip families of transcription factors in regulating hRFC expression.
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Papers by Johnathan Whetstine