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1.  Epigenetic and genetic inactivation of tyrosyl-DNA-phosphodiesterase 1 (TDP1) in human lung cancer cells from the NCI-60 panel 
DNA repair  2013;13:1-9.
Tyrosyl-DNA-phosphodiesterase 1 (TDP1) repairs 3’-blocking DNA lesions by catalytically hydrolyzing the tyrosyl-DNA-phosphodiester bond of trapped topoisomerase I (Top1) cleavage complexes (Top1cc). It also removes 3’-blocking residues derived from oxidative damage or incorporation of chain terminating anticancer and antiviral nucleosides. Thus, TDP1 is regarded as a determinant of resistance to Top1 inhibitors and chain terminating nucleosides, and possibly of genomic stability. In the 60 cell lines of the NCI Developmental Therapeutic Anticancer Screen (the NCI-60), whose whole genome transcriptome and mutations have recently been characterized, we discovered two human lung cancer cell lines deficient for TDP1 (NCI_H522 and HOP_62). HOP_62 shows undetectable TDP1 mRNA and NCI_H522 bears a homozygous deleterious mutation of TDP1 at a highly conserved amino acid residue (K292E). Absence of TDP1 protein and lack of TDP1 catalytic activity were demonstrated in cell lysates from both cell lines. Lack of TDP1 expression in HOP_62 was shown to be due to TDP1 promoter hypermethylation. Our study provides insights into the possible inactivation of TDP1 in cancers and its relationship to cellular response to Top1-targeted drugs. It also reveals two TDP1 knockout lung cancer cell lines for further TDP1 functional analyses.
doi:10.1016/j.dnarep.2013.09.001
PMCID: PMC3919147  PMID: 24355542
TDP1; Topoisomerases; topotecan; CellMiner; promoter hypermethylation
2.  Nucleosomes are enriched at the boundaries of hypomethylated regions (HMRs) in mouse dermal fibroblasts and keratinocytes 
Epigenetics & Chromatin  2014;7(1):34.
Background
The interplay between epigenetic modifications and chromatin structure are integral to our understanding of genome function. Methylation of cytosine (5mC) at CG dinucleotides, traditionally associated with transcriptional repression, is the most highly studied chemical modification of DNA, occurring at over 70% of all CG dinucleotides in the genome. Hypomethylated regions (HMRs) often occur in CG islands (CGIs), however, they also occur outside of CGIs and function as cell-type specific enhancers. During the process of differentiation, reorganization of chromatin and nucleosome arrangement at regulatory regions is thought to occur in order for the establishment of cell-type specific transcriptional programs. However, the specifics regarding the organization of nucleosomes at HMRs and the potential mechanisms regulating nucleosome occupancy in these regions are unknown. Here, we have investigated nucleosome organization around hypomethylated regions (HMRs) identified in two mouse primary cells.
Results
Microccocal nuclease (MNase) digested mononucleosomes from primary cultures of new-born female mouse dermal fibroblasts and keratinocytes were mapped and compared to the HMRs obtained from single base-pair resolution methylomes. In both cell types, we find that nucleosomes are enriched at HMR boundaries. In contrast to the nucleosomes found at boundaries of HMRs in CGIs, HMRs outside of CGIs are calculated to be preferentially bound by nucleosomes, with phased nucleosomes propagating into the methylated region. Nucleosomes are enriched at the tissue-specific HMRs (TS-HMR) boundaries in both cell types suggesting that nucleosome organization surrounding HMR boundaries is independent of methylation status. In addition, we find potential transcription factor (TF) binding sites (E-box motifs) enriched in non-CGI TS-HMR boundaries.
Conclusions
Our results show that intrinsic nucleosome occupancy score (INOS) positively correlate with the nucleosome organization surrounding non-CGI TS-HMRs, suggesting that DNA sequence plays a role in the establishment of HMRs in the genome. Since nucleosomes impact all processes involving the genome, our results provide a link between epigenetic modifications, chromatin structure, and regulatory function.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-8935-7-34) contains supplementary material, which is available to authorized users.
doi:10.1186/1756-8935-7-34
PMCID: PMC4265496  PMID: 25506399
CG methylation; Hypomethylated regions; HMR; Nucleosomes; Epigenomics; Keratinocytes; Fibroblasts
3.  High-resolution genome-wide DNA methylation maps of mouse primary female dermal fibroblasts and keratinocytes 
Background
Genome-wide DNA methylation at a single nucleotide resolution in different primary cells of the mammalian genome helps to determine the characteristics and functions of tissue-specific hypomethylated regions (TS-HMRs). We determined genome-wide cytosine methylation maps at 91X and 36X coverage of newborn female mouse primary dermal fibroblasts and keratinocytes and compared with mRNA-seq gene expression data.
Results
These high coverage methylation maps were used to identify HMRs in both cell types. A total of 2.91% of the genome are in keratinocyte HMRs, and 2.15% of the genome are in fibroblast HMRs with 1.75% being common. Half of the TS-HMRs are extensions of common HMRs, and the remaining are unique TS-HMRs. Four levels of CG methylation are observed: 1) total unmethylation for CG dinucleotides in HMRs in CGIs that are active in all tissues; 2) 10% to 40% methylation for TS-HMRs; 3) 60% methylation for TS-HMRs in cells types where they are not in HMRs; and 4) 70% methylation for the nonfunctioning part of the genome. SINE elements are depleted inside the TS-HMRs, while highly enriched in the surrounding regions. Hypomethylation at the last exon shows gene repression, while demethylation toward the gene body positively correlates with gene expression. The overlapping HMRs have a more complex relationship with gene expression. The common HMRs and TS-HMRs are each enriched for distinct Transcription Factor Binding Sites (TFBS). C/EBPβ binds to methylated regions outside of HMRs while CTCF prefers to bind in HMRs, highlighting these two parts of the genome and their potential interactions.
Conclusions
Keratinocytes and fibroblasts are of epithelial and mesenchymal origin. High-resolution methylation maps in these two cell types can be used as reference methylomes for analyzing epigenetic mechanisms in several diseases including cancer.
Please see related article at the following link: http://www.epigeneticsandchromatin.com/content/7/1/34
Electronic supplementary material
The online version of this article (doi:10.1186/1756-8935-7-35) contains supplementary material, which is available to authorized users.
doi:10.1186/1756-8935-7-35
PMCID: PMC4333159
CG methylation; Hypomethylated regions; HMR; Methylome; CTCF; C/EBPβ; Keratinocytes; Fibroblasts
4.  Fabrication of duplex DNA microarrays incorporating methyl-5-cytosine 
Lab on a chip  2011;12(2):376-380.
We synthesized customized double-stranded DNA microarrays including methyl-5-cytosine at CpG dinucleotides and produced all 163,555 possible 8-mers (un-, hemi-, and di-methylated) to gain insight into how methylation affects transcription factor binding. An antibody to methyl-5-cytidine showed greater binding to the methylated DNA, demonstrating efficient incorporation of methyl-5-cytosine into the synthesized DNA. In contrast, binding of the transcription factor CREB was inhibited by CpG methylation. This platform represents a powerful new technology to evaluate the effect of DNA methylation on protein binding in any sequence context.
doi:10.1039/c1lc20698b
PMCID: PMC4205155  PMID: 22139143
5.  A Calcium-Initiated Signaling Pathway Propagated through Calcineurin and cAMP Response Element-Binding Protein Activates Proenkephalin Gene Transcription after Depolarization 
Molecular pharmacology  2003;64(6):1503-1511.
Essential components of a signal-transduction pathway regulating activity-dependent neuropeptide gene transcription have been identified. Proenkephalin (PEnk) gene activation after depolarization of chromaffin cells with 40 mM KCl was blocked by the voltage-sensitive calcium-channel blocker methoxyverapamil (D600) (30 μM) and by calcineurin inhibition with 100 nM cyclosporin A or ascomycin but not by inhibiting new protein synthesis with 0.5 μg/ml cycloheximide. KCl-induced elevation of PEnk mRNA was distinct from activation of the PEnk gene by either cAMP or protein kinase C. Twenty-five micromolar forskolin- and 100 nM phorbol 12-myristate 13-acetate-induced elevations of PEnk mRNA were cycloheximide-sensitive and were not blocked by cyclosporin A or ascomycin. KCl stimulated Ser-133 phosphorylation of cAMP response element-binding protein (CREB) in chromaffin cells, and CREB phosphorylation was blocked by both ascomycin and D600. A reporter gene containing 193 bases of the PEnk gene 5′ flank driving luciferase gene expression (pENK12-Luc) transfected into chromaffin cells was transcriptionally activated by KCl depolarization. Activation was blocked by both ascomycin and D600 and required an intact CREB binding site (ENKCRE2). An oligonucleotide containing the PEnk cAMP response element-2 was gel-shifted by both unstimulated and potassium-stimulated chromaffin cell nuclear extracts into a prominent complex supershifted by CREB antibodies. Finally, stimulation of transcription of the pENK12-Luc reporter by KCl in chromaffin cells was blocked by coexpression of the CREB antagonist A-CREB but not by the AP-1 antagonist A-Fos. Stimulus-transcription coupling after depolarization in chromaffin cells occurs via calcineurin-dependent activation of CREB, a pathway distinct from cAMP- or protein kinase C-initiated signaling and independent of immediate early gene regulation.
doi:10.1124/mol.64.6.1503
PMCID: PMC4186738  PMID: 14645681
6.  Correction: Combinatorial Recruitment of CREB, C/EBPβ and c-Jun Determines Activation of Promoters upon Keratinocyte Differentiation 
PLoS ONE  2013;8(12):10.1371/annotation/0cd737c6-949b-48f9-8bc8-ab36376c03e3.
doi:10.1371/annotation/0cd737c6-949b-48f9-8bc8-ab36376c03e3
PMCID: PMC3878901
7.  Combinatorial Recruitment of CREB, C/EBPβ and c-Jun Determines Activation of Promoters upon Keratinocyte Differentiation 
PLoS ONE  2013;8(11):e78179.
Background
Transcription factors CREB, C/EBPβ and Jun regulate genes involved in keratinocyte proliferation and differentiation. We questioned if specific combinations of CREB, C/EBPβ and c-Jun bound to promoters correlate with RNA polymerase II binding, mRNA transcript levels and methylation of promoters in proliferating and differentiating keratinocytes.
Results
Induction of mRNA and RNA polymerase II by differentiation is highest when promoters are bound by C/EBP β alone, C/EBPβ together with c-Jun, or by CREB, C/EBPβ and c-Jun, although in this case CREB binds with low affinity. In contrast, RNA polymerase II binding and mRNA levels change the least upon differentiation when promoters are bound by CREB either alone or in combination with C/EBPβ or c-Jun. Notably, promoters bound by CREB have relatively high levels of RNA polymerase II binding irrespective of differentiation. Inhibition of C/EBPβ or c-Jun preferentially represses mRNA when gene promoters are bound by corresponding transcription factors and not CREB. Methylated promoters have relatively low CREB binding and, accordingly, those which are bound by C/EBPβ are induced by differentiation irrespective of CREB. Composite “Half and Half” consensus motifs and co localizing consensus DNA binding motifs are overrepresented in promoters bound by the combination of corresponding transcription factors.
Conclusion
Correlational and functional data describes combinatorial mechanisms regulating the activation of promoters. Colocalization of C/EBPβ and c-Jun on promoters without strong CREB binding determines high probability of activation upon keratinocyte differentiation.
doi:10.1371/journal.pone.0078179
PMCID: PMC3820678  PMID: 24244291
8.  Inhibition of Leptin Regulation of Parasympathetic Signaling as a Cause of Extreme Body Weight-Associated Asthma 
Cell metabolism  2013;17(1):10.1016/j.cmet.2012.12.004.
SUMMARY
Impaired lung function caused by decreased airway diameter (bronchoconstriction) is frequently observed whether body weight is abnormally high or low. That these opposite conditions affect the airways similarly suggests that the regulation of airway diameter and body weight are intertwined. We show here that, independently of its regulation of appetite, melanocortin pathway, or sympathetic tone, leptin is necessary and sufficient to increase airway diameter by signaling through its cognate receptor in cholinergic neurons. The latter decreases parasympathetic signaling through the M3 muscarinic receptor in airway smooth muscle cells, thereby increasing airway diameter without affecting local inflammation. Accordingly, decreasing parasympathetic tone genetically or pharmacologically corrects bronchoconstriction and normalizes lung function in obese mice regardless of bronchial inflammation. This study reveals an adipocyte-dependent regulation of bronchial diameter whose disruption contributes to the impaired lung function caused by abnormal body weight. These findings may be of use in the management of obesity-associated asthma.
doi:10.1016/j.cmet.2012.12.004
PMCID: PMC3815545  PMID: 23312282
9.  CG methylation 
Epigenomics  2012;4(6):655-663.
A striking feature of mammalian genomes is the paucity of the CG dinucleotide. There are approximately 20,000 regions termed CpG islands where CGs cluster. This represents 5% of all CGs and 1% of the genome. CpG islands are typically unmethylated and are often promoters for housekeeping genes. The remaining 95% of CG dinucleotides are disposed throughout 99% of the genome and are typically methylated and found in half of all promoters. CG methylation facilitates binding of the C/EBP family of transcription factors, proteins critical for differentiation of many tissues. This allows these proteins to localize in the methylated CG poor regions of the genome where they may produce advantageous changes in gene expression at nearby or more distant regions of the genome. In this review, our growing understanding of the consequences of CG methylation will be surveyed.
doi:10.2217/epi.12.55
PMCID: PMC3566568  PMID: 23244310
C/EBP; CG; CG dinucleotide; CGI; CpG; CpG island; cytosine; methylation; TFBS; tissue specific
10.  CpG methylation recruits sequence specific transcription factors essential for tissue specific gene expression 
Biochimica et Biophysica Acta  2012;1819(7):763-770.
CG methylation is an epigenetically inherited chemical modification of DNA found in plants and animals. In mammals it is essential for accurate regulation of gene expression and normal development. Mammalian genomes are depleted for the CG dinucleotide, a result of the chemical deamination of methyl-cytosine in CG resulting in TpG. Most CG dinucleotides are methylated, but ~ 15% are unmethylated. Five percent of CGs cluster into ~20,000 regions termed CG islands (CGI) which are generally unmethylated. About half of CGIs are associated with housekeeping genes. In contrast, the gene body, repeats and transposable elements in which CGs are generally methylated. Unraveling the epigenetic machinery operating in normal cells is important for understanding the epigenetic aberrations that are involved in human diseases including cancer. With the advent of high-throughput sequencing technologies, it is possible to identify the CG methylation status of all 30 million unique CGs in the human genome, and monitor differences in distinct cell types during differentiation and development. Here we summarize the present understanding of DNA methylation in normal cells and discuss resent observations that CG methylation can have an effect on tissue specific gene expression. We also discuss how aberrant CG methylation can lead to cancer.
doi:10.1016/j.bbagrm.2012.02.014
PMCID: PMC3371161  PMID: 22387149
Epigenetics; DNA methylation; transcription; CpG Island; Cancer; 5-azacytidine
11.  Contribution of nucleosome binding preferences and co-occurring DNA sequences to transcription factor binding 
BMC Genomics  2013;14:428.
Background
Chromatin plays a critical role in regulating transcription factors (TFs) binding to their canonical transcription factor binding sites (TFBS). Recent studies in vertebrates show that many TFs preferentially bind to genomic regions that are well bound by nucleosomes in vitro. Co-occurring secondary motifs sometimes correlated with functional TFBS.
Results
We used a logistic regression to evaluate how well the propensity for nucleosome binding and co-occurrence of a secondary motif identify which canonical motifs are bound in vivo. We used ChIP-seq data for three transcription factors binding to their canonical motifs: c-Jun binding the AP-1 motif (TGAC/GTCA), GR (glucocorticoid receptor) binding the GR motif (G-ACA---T/CGT-C), and Hoxa2 (homeobox a2) binding the Pbx (Pre-B-cell leukemia homeobox) motif (TGATTGAT). For all canonical TFBS in the mouse genome, we calculated intrinsic nucleosome occupancy scores (INOS) for its surrounding 150-bps DNA and examined the relationship with in vivo TF binding. In mouse mammary 3134 cells, c-Jun and GR proteins preferentially bound regions calculated to be well-bound by nucleosomes in vitro with the canonical AP-1 and GR motifs themselves contributing to the high INOS. Functional GR motifs are enriched for AP-1 motifs if they are within a nucleosome-sized 150-bps region. GR and Hoxa2 also bind motifs with low INOS, perhaps indicating a different mechanism of action.
Conclusion
Our analysis quantified the contribution of INOS and co-occurring sequence to the identification of functional canonical motifs in the genome. This analysis revealed an inherent competition between some TFs and nucleosomes for binding canonical TFBS. GR and c-Jun cooperate if they are within 150-bps. Binding of Hoxa2 and a fraction of GR to motifs with low INOS values suggesting they are not in competition with nucleosomes and may function using different mechanisms.
doi:10.1186/1471-2164-14-428
PMCID: PMC3700821  PMID: 23805837
TFBS; Nucleosome; GR; c-Jun
12.  Deficiency of CCAAT/enhancer binding protein family DNA binding prevents malignant conversion of adenoma to carcinoma in NNK-induced lung carcinogenesis in the mouse 
Molecular Cancer  2012;11:90.
Background
The CCAAT/enhancer binding proteins (C/EBPs) play important roles in carcinogenesis of many tumors including the lung. Since multiple C/EBPs are expressed in lung, the combinatorial expression of these C/EBPs on lung carcinogenesis is not known.
Methods
A transgenic mouse line expressing a dominant negative A-C/EBP under the promoter of lung epithelial Clara cell secretory protein (CCSP) gene in doxycycline dependent fashion was subjected to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis bioassay in the presence and absence of doxycycline, and the effect of abolition of DNA binding activities of C/EBPs on lung carcinogenesis was examined.
Results
A-C/EBP expression was found not to interfere with tumor development; however, it suppressed the malignant conversion of adenoma to carcinoma during NNK-induced lung carcinogenesis. The results suggested that Ki67 may be used as a marker for lung carcinomas in mouse.
Conclusions
The DNA binding of C/EBP family members can be used as a potential molecular target for lung cancer therapy.
doi:10.1186/1476-4598-11-90
PMCID: PMC3548712  PMID: 23234329
C/EBPs; Lung chemical carcinogenesis bioassay; Dominant negative; A-C/EBP; Transgenic mouse; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNK
13.  Overlapping ETS and CRE Motifs (G/CCGGAAGTGACGTCA) Preferentially Bound by GABPα and CREB Proteins 
G3: Genes|Genomes|Genetics  2012;2(10):1243-1256.
Previously, we identified 8-bps long DNA sequences (8-mers) that localize in human proximal promoters and grouped them into known transcription factor binding sites (TFBS). We now examine split 8-mers consisting of two 4-mers separated by 1-bp to 30-bps (X4-N1-30-X4) to identify pairs of TFBS that localize in proximal promoters at a precise distance. These include two overlapping TFBS: the ETS⇔ETS motif (C/GCCGGAAGCGGAA) and the ETS⇔CRE motif (C/GCGGAAGTGACGTCAC). The nucleotides in bold are part of both TFBS. Molecular modeling shows that the ETS⇔CRE motif can be bound simultaneously by both the ETS and the B-ZIP domains without protein-protein clashes. The electrophoretic mobility shift assay (EMSA) shows that the ETS protein GABPα and the B-ZIP protein CREB preferentially bind to the ETS⇔CRE motif only when the two TFBS overlap precisely. In contrast, the ETS domain of ETV5 and CREB interfere with each other for binding the ETS⇔CRE. The 11-mer (CGGAAGTGACG), the conserved part of the ETS⇔CRE motif, occurs 226 times in the human genome and 83% are in known regulatory regions. In vivo GABPα and CREB ChIP-seq peaks identified the ETS⇔CRE as the most enriched motif occurring in promoters of genes involved in mRNA processing, cellular catabolic processes, and stress response, suggesting that a specific class of genes is regulated by this composite motif.
doi:10.1534/g3.112.004002
PMCID: PMC3464117  PMID: 23050235
proximal promoters; transcription factor binding sites; co-localization; transcriptional start site; EMSA
14.  The Biology of Tobacco and Nicotine: Bench to Bedside 
Strong epidemiologic evidence links smoking and cancer. An increased understanding of the molecular biology of tobacco-related cancers could advance progress toward improving smoking cessation and patient management. Knowledge gaps between tobacco addiction, tumorigenesis, and cancer brought an interdisciplinary group of investigators together to discuss “The Biology of Nicotine and Tobacco: Bench to Bedside.” Presentations on the signaling pathways and pathogenesis in tobacco-related cancers, mouse models of addiction, imaging and regulation of nicotinic receptors, the genetic basis for tobacco carcinogenesis and development of lung cancer, and molecular mechanisms of carcinogenesis were heard. Importantly, new opportunities to use molecular biology to identify and abrogate tobacco-mediated carcinogenesis and to identify high-risk individuals were recognized.
doi:10.1158/1055-9965.EPI-04-0652
PMCID: PMC3459058  PMID: 15824140
15.  Transcription Factor AP1 Potentiates Chromatin Accessibility and Glucocorticoid Receptor Binding 
Molecular cell  2011;43(1):145-155.
Summary
Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with co-regulators. The role of these interactions in determining selective binding of GR to regulatory elements remains unclear. Recent findings indicate a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing, we identify the activator protein 1 (AP1) as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. We propose a model where the basal occupancy of transcription factors act to prime chromatin and direct inducible transcription factors to select regions in the genome.
doi:10.1016/j.molcel.2011.06.016
PMCID: PMC3138120  PMID: 21726817
16.  Suppression of the C/EBP family of transcription factors in adipose tissue causes lipodystrophy 
Adipose-specific inactivation of both AP-1 and C/EBP families of B-ZIP transcription factors in transgenic mice causes severe lipoatrophy. To evaluate if inactivation of only C/EBP members was critical for lipoatrophy, A-C/EBP, a dominant-negative protein that specifically inhibits the DNA binding of the C/EBP members, was expressed in adipose tissue. For first 2 weeks after birth, aP2-A-C/EBP mice had no white adipose tissue(WAT), drastically reduced brown adipose tissue(BAT) and exhibited marked hepatic steatosis, hyperinsulinemia, and hyperlipidemia. However, WAT appeared during the third week, coinciding with significantly improved metabolic functioning. In adults, BAT remained reduced, causing cold intolerance. At 30 weeks, the aP2-A-C/EBP mice had only 35% reduced WAT, with clear morphological signs of lipodystrophy in subcutaneous fat. Circulating leptin and adiponectin levels were less than the wild type levels and these mice exhibited impaired triglyceride clearance. Insulin resistance, glucose intolerance, and reduced free fatty acid release in response to β3-adrenergic agonist suggest improper functioning of the residual WAT. Gene-expression analysis of inguinal WAT identified reduced mRNA levels of several enzymes involved in fatty acid synthesis and glucose metabolism that are known C/EBPα transcriptional targets. There were increased levels for genes involved in inflammation and muscle differentiation. However, when dermal-fibroblasts from aP2-A-C/EBP mice were differentiated into adipocytes in tissue culture, muscle markers were elevated more than the inflammatory markers. These results demonstrate that the C/EBP family is essential for adipose tissue development during the early postnatal period, contribute to glucose and lipid homeostasis in adults, and the suppression of the muscle lineage.
doi:10.1530/JME-10-0172
PMCID: PMC3159190  PMID: 21321096
C/EBP; dominant negative; adipose tissue; diabetes; lipodystrophy; transgenic mouse
17.  Predisposition to Cancer Caused by Genetic and Functional Defects of Mammalian Atad5 
PLoS Genetics  2011;7(8):e1002245.
ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5+/m) mice that were haploinsuffficient for Atad5. Atad5+/m mice displayed high levels of genomic instability in vivo, and Atad5+/m mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5+/m mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5+/m mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis.
Author Summary
Genomic instability is a hallmark of tumorigenesis, suggesting that mutations in genes suppressing genomic instability contribute to this phenotype. In this study, we demonstrate for the first time that haploinsufficiency for Atad5, a protein that is important in stabilizing stalled DNA replication forks by regulating PCNA ubiquitination during DNA damage bypass, predisposes >90% of mice to tumorigenesis in multiple organs. In heterozygous Atad5 mice, both somatic cells and the spontaneous tumors showed high levels of genomic instability. In a subset of sporadic human endometrial tumors, we identified heterozygous loss-of-function somatic mutations in the ATAD5 gene, consistent with the role of mouse Atad5 in suppressing tumorigenesis. Collectively, our findings suggest that ATAD5 may be a novel tumor suppressor gene.
doi:10.1371/journal.pgen.1002245
PMCID: PMC3161924  PMID: 21901109
18.  Arxes: retrotransposed genes required for adipogenesis 
Nucleic Acids Research  2010;39(8):3224-3239.
Retrotransposed sequences arise from messenger RNAs (mRNAs) that have been reinserted into genomic DNA by reverse transcription. Usually, these sequences are embedded in dormant regions, collect missense mutations over time and constitute processed, nonfunctional pseudogenes. There are thousands of processed pseudogenes in the mouse and human genome. Here, we report evidence for two paralog genes (termed Arxes1 and Arxes2), which arose by retrotransposition of the signal peptidase Spcs3 followed by a segmental duplication event. They gained a functional promoter that we show to be transactivated by adipogenic transcription factors. We further show that the Arxes mRNAs are highly expressed in adipose tissue and strongly upregulated during adipogenesis in different cell models. Additionally, their expression is elevated by an anti-diabetic agent in vitro and in vivo. Importantly, we provide evidence that the Arxes genes are translated and that the proteins are located in the endoplasmic reticulum. Although the sequence similarity and subcellular location are reminiscent of their parental gene, our data suggest that the Arxes have developed a different function, since their expression is required for adipogenesis, whereas Spcs3 is dispensable. In summary, we report retrotransposed-duplicated genes that evolved from a parental gene to function in a tissue and adipogenesis-specific context.
doi:10.1093/nar/gkq1289
PMCID: PMC3082915  PMID: 21177646
19.  Opposing Roles for ATF2 and c-Fos in c-Jun-Mediated Neuronal Apoptosis▿  
Molecular and Cellular Biology  2009;29(9):2431-2442.
The activator protein 1 (AP-1) transcription factor c-Jun is crucial for neuronal apoptosis. However, c-Jun dimerization partners and the regulation of these proteins in neuronal apoptosis remain unknown. Here we report that c-Jun-mediated neuronal apoptosis requires the concomitant activation of activating transcription factor-2 (ATF2) and downregulation of c-Fos. Furthermore, we have observed that c-Jun predominantly heterodimerizes with ATF2 and that the c-Jun/ATF2 complex promotes apoptosis by triggering ATF activity. Inhibition of c-Jun/ATF2 heterodimerization using dominant negative mutants, small hairpin RNAs, or decoy oligonucleotides was able to rescue neurons from apoptosis, whereas constitutively active ATF2 and c-Jun mutants were found to synergistically stimulate apoptosis. Bimolecular fluorescence complementation analysis confirmed that, in living neurons, c-Fos downregulation facilitates c-Jun/ATF2 heterodimerization. A chromatin immunoprecipitation assay also revealed that c-Fos expression prevents the binding of c-Jun/ATF2 heterodimers to conserved ATF sites. Moreover, the presence of c-Fos is able to suppress the expression of c-Jun/ATF2-mediated target genes and, therefore, apoptosis. Taken together, our findings provide evidence that potassium deprivation-induced neuronal apoptosis is mediated by concurrent upregulation of c-Jun/ATF2 heterodimerization and downregulation of c-Fos expression. This paradigm demonstrates opposing roles for ATF2 and c-Fos in c-Jun-mediated neuronal apoptosis.
doi:10.1128/MCB.01344-08
PMCID: PMC2668374  PMID: 19255142
20.  5′UTR of the Neurogenic bHLH Nex1/MATH-2/NeuroD6 Gene Is Regulated by Two Distinct Promoters Through CRE and C/EBP Binding Sites 
Expression of the bHLH transcription factor Nex1/MATH-2/NeuroD6, a member of the NeuroD subfamily, parallels overt neuronal differentiation and synaptogenesis during brain development. Our previous studies have shown that Nex1 is a critical effector of the NGF pathway and promotes neuronal differentiation and survival of PC12 cells in the absence of growth factors. In this study, we investigated the transcriptional regulation of the Nex1 gene during NGF-induced neuronal differentiation. We found that Nex1 expression is under the control of two conserved promoters, Nex1-P1 and Nex1-P2, located in two distinct non-coding exons. Both promoters are TATA-less with multiple transcription start sites, and are activated on NGF or cAMP exposure. Luciferase-reporter assays showed that the Nex1-P2 promoter activity is stronger than the Nex1-P1 promoter activity, which supports the previously reported differential expression levels of Nex1 transcripts throughout brain development. Using a combination of DNaseI footprinting, EMSA assays, and site-directed mutagenesis, we identified the essential regulatory elements within the first 2 kb of the Nex1 5′UTR. The Nex1-P1 promoter is mainly regulated by a conserved CRE element, whereas the Nex1-P2 promoter is under the control of a conserved C/EBP binding site. Overexpression of wild-type C/EBPβ resulted in increased Nex1-P2 promoter activity in NGF-differentiated PC12 cells. The fact that Nex1 is a target gene of C/EBPβ provides new insight into the C/EBP transcriptional cascade known to promote neurogenesis, while repressing gliogenesis.
doi:10.1002/jnr.21093
PMCID: PMC2767119  PMID: 17075921
neuronal differentiation; bHLH transcription factor; C/EBP; CREB; NeuroD family
21.  A combined yeast/bacterial two-hybrid system (YBTH): development and evaluation 
Summary
Two-hybrid screening is a standard methodology to identify and characterize protein-protein interactions that has become an integral component of many proteomic investigations. The two-hybrid system was initially developed using yeast as a host organism. However, bacterial two-hybrid systems have also become common laboratory tools and are preferred in some circumstances, although yeast and bacterial two-hybrid systems have never been directly compared. We describe here the development of a unified yeast and bacterial two-hybrid (YBTH) system in which a single bait expression plasmid is used in both organismal milieus. We use a series of leucine zipper fusion proteins of known affinities to compare interaction detection using both systems. While both two-hybrid systems detected interactions within a comparable range of interaction affinities, each demonstrated unique advantages. The yeast system produced quantitative readout over a greater dynamic range than that observed with bacteria. However, the phenomenon of “auto-activation” by baits was less problematic in the bacterial system than in yeast. Both systems identified physiological interactors for a library screen with a cI-Ras test bait; however, non-identical interactors were obtained in yeast and bacterial screens. The ability to rapidly shift between yeast and bacterial systems provided by these new reagents should provide a marked advantage for two-hybrid investigations. In addition, the modified expression vectors should be useful for any application requiring facile expression of a protein of interest in both yeast and bacteria.
doi:10.1074/mcp.T500005-MCP200
PMCID: PMC2670611  PMID: 15781424
Two-hybrid; protein-protein interaction; leucine zipper; Ras; bacteria; yeast
22.  Inhibiting AP-1 activity alters cocaine induced gene expression and potentiates sensitization 
Neuroscience  2008;152(4):1040-1053.
We have expressed A-FOS, an inhibitor of AP-1 DNA binding, in adult mouse striatal neurons. We observe normal behavior including locomotion and exploratory activities. Following a single injection of cocaine, locomotion increased similarly in both the A-FOS expressing and littermate controls. However, following repeated injections of cocaine, the A-FOS expressing mice showed increased locomotion relative to littermate controls, an increase that persisted following a week of withdrawal and subsequent cocaine administration. These results indicate that AP-1 suppresses this behavioral responses to cocaine. We analyzed mRNA from the striatum before and 4 and 24 hours after a single cocaine injection in both A-FOS and control striata using Affymetrix microarrays (430 2.0 Array) to identify genes mis-regulated by A-FOS that may mediate the increased locomotor sensitization to cocaine. A-FOS expression did not change gene expression in the basal state or 4 hours following cocaine treatment relative to controls. However, 24 hours after an acute cocaine treatment, 84 genes were identified that were differentially expressed between the A-FOS and control mice. 56 gene are down regulated while 28 genes are up regulated including previously identified candidates for addiction including BDNF and Per1. Using a random sample of identified genes, quantitative PCR was used to verify the microarray studies. The chromosomal location of these 84 genes was compared to human genome scans of addiction to identify potential genes in humans that are involved in addiction.
doi:10.1016/j.neuroscience.2008.01.045
PMCID: PMC2585517  PMID: 18355967
A-FOS; addiction; sensitization; gene expression
23.  All and only CpG containing sequences are enriched in promoters abundantly bound by RNA polymerase II in multiple tissues 
BMC Genomics  2008;9:67.
Background
The promoters of housekeeping genes are well-bound by RNA polymerase II (RNAP) in different tissues. Although the promoters of these genes are known to contain CpG islands, the specific DNA sequences that are associated with high RNAP binding to housekeeping promoters has not been described.
Results
ChIP-chip experiments from three mouse tissues, liver, heart ventricles, and primary keratinocytes, indicate that 94% of promoters have similar RNAP binding, ranging from well-bound to poorly-bound in all tissues. Using all 8-base pair long sequences as a test set, we have identified the DNA sequences that are enriched in promoters of housekeeping genes, focusing on those DNA sequences which are preferentially localized in the proximal promoter. We observe a bimodal distribution. Virtually all sequences enriched in promoters with high RNAP binding values contain a CpG dinucleotide. These results suggest that only transcription factor binding sites (TFBS) that contain the CpG dinucleotide are involved in RNAP binding to housekeeping promoters while TFBS that do not contain a CpG are involved in regulated promoter activity. Abundant 8-mers that are preferentially localized in the proximal promoters and exhibit the best enrichment in RNAP bound promoters are all variants of six known CpG-containing TFBS: ETS, NRF-1, BoxA, SP1, CRE, and E-Box. The frequency of these six DNA motifs can predict housekeeping promoters as accurately as the presence of a CpG island, suggesting that they are the structural elements critical for CpG island function. Experimental EMSA results demonstrate that methylation of the CpG in the ETS, NRF-1, and SP1 motifs prevent DNA binding in nuclear extracts in both keratinocytes and liver.
Conclusion
In general, TFBS that do not contain a CpG are involved in regulated gene expression while TFBS that contain a CpG are involved in constitutive gene expression with some CpG containing sequences also involved in inducible and tissue specific gene regulation. These TFBS are not bound when the CpG is methylated. Unmethylated CpG dinucleotides in the TFBS in CpG islands allow the transcription factors to find their binding sites which occur only in promoters, in turn localizing RNAP to promoters.
doi:10.1186/1471-2164-9-67
PMCID: PMC2267717  PMID: 18252004
24.  Refined Spatial Manipulation of Neuronal Function by Combinatorial Restriction of Transgene Expression 
Neuron  2006;52(3):425-436.
Summary
Selective genetic manipulation of neuronal function in vivo requires techniques for targeting gene expression to specific cells. Existing systems accomplish this using the promoters of endogenous genes to drive expression of transgenes directly in cells of interest, or, in “binary” systems, to drive expresssion of a transcription factor or recombinase that subsequently activates the expression of other transgenes. All such techniques are constrained by the limited specificity of the available promoters. We introduce here a combinatorial system in which the DNA-binding (DBD) and transcription activation (AD) domains of a transcription factor are independently targeted using two different promoters. The domains heterodimerize to become transcriptionally competent and thus drive trangene expression only at the intersection of the expression patterns of the two promoters. We use this system to dissect a neuronal network in Drosophila by selectively targeting expression of the cell death gene reaper to subsets of neurons within the network.
doi:10.1016/j.neuron.2006.08.028
PMCID: PMC1713190  PMID: 17088209
Gene Targeting; Transgenic; Gal4; Neural Network; Circuit; Drosophila
25.  Math6 expression during kidney development and altered expression in a mouse model of glomerulosclerosis 
Math6 is a tissue-restricted member of the Atonal family of bHLH transcription factors and has been implicated in specification and differentiation of cell lineages in the brain. We identify here Math6 as a podocyte expressed bHLH protein that was downregulated in HIV-associated nephropathy; a collapsing glomerulopathy characterized by podocyte dedifferentiation. Early in metanephric development, Math6 was expressed in metanephric mesenchyme, but not ureteric bud-derived cells, with overall Math6 expression most abundant in the nephrogenic zone, including developing glomeruli. In adult kidney, Math6 expression was restricted to podocytes. In adult podocyte cell lines and kidneys from the transgenic mouse model of HIVAN, Math6 podocyte expression was reduced concurrent with previously reported reductions in Nephrin and Synaptopodin expression, suggesting a correlation between the loss of Math6 expression and typical podocyte terminal differentiation markers. These studies suggest that Math6 may participate in kidney development, and may be a permissive factor for podocyte differentiation.
doi:10.1002/dvdy.20934
PMCID: PMC2203212  PMID: 16937370
podocyte; HIV-associated nephropathy; HIV-1; transcriptional regulation grant support: NIH DK61395

Results 1-25 (37)