Catharine L Smith

Interests

Research

Steroid receptors, lysine deacetylases, transcription, lymphoma treatment, targeting the epigenome

Teaching

Nuclear Receptors, Transcription, Acetylation, Cancer, Metabolism

Courses

Scholarly Contributions

Journals/Publications

• Griggs, C. A., Malm, S. W., Jaime-Frias, R., & Smith, C. L. (2018). Valproic acid disrupts the oscillatory expression of core circadian rhythm transcription factors. Toxicology and applied pharmacology, 339, 110-120.
  More info

  Valproic acid (VPA) is a well-established therapeutic used in treatment of seizure and mood disorders as well as migraines and a known hepatotoxicant. About 50% of VPA users experience metabolic disruptions, including weight gain, hyperlipidemia, and hyperinsulinemia, among others. Several of these metabolic abnormalities are similar to the effects of circadian rhythm disruption. In the current study, we examine the effect of VPA exposure on the expression of core circadian transcription factors that drive the circadian clock via a transcription-translation feedback loop. In cells with an unsynchronized clock, VPA simultaneously upregulated the expression of genes encoding core circadian transcription factors that regulate the positive and negative limbs of the feedback loop. Using low dose glucocorticoid, we synchronized cultured fibroblast cells to a circadian oscillatory pattern. Whether VPA was added at the time of synchronization or 12h later at CT12, we found that VPA disrupted the oscillatory expression of multiple genes encoding essential transcription factors that regulate circadian rhythm. Therefore, we conclude that VPA has a potent effect on the circadian rhythm transcription-translation feedback loop that may be linked to negative VPA side effects in humans. Furthermore, our study suggests potential chronopharmacology implications of VPA usage.
• Patrick, N. M., Griggs, C. A., Icenogle, A. L., Gilpatrick, M. M., Kadiyala, V., Jaime-Frias, R., & Smith, C. L. (2017). Class I lysine deacetylases promote glucocorticoid-induced transcriptional repression through functional interaction with LSD1. The Journal of steroid biochemistry and molecular biology, 167, 1-13.
  More info

  Small molecule inhibitors of lysine deacetylases (KDACs) are approved for clinical use in treatment of several diseases. Nuclear receptors, such as the glucocorticoid receptor (GR) use lysine acetyltransferases (KATs or HATs) and KDACs to regulate transcription through acetylation and deacetylation of protein targets such as histones. Previously we have shown that KDAC1 activity facilitates GR-activated transcription at about half of all cellular target genes. In the current study we examine the role of Class I KDACs in glucocorticoid-mediated repression of gene expression. Inhibition of KDACs through two structurally distinct Class I-selective inhibitors prevented dexamethasone (Dex)-mediated transcriptional repression in a gene-selective fashion. In addition, KDAC activity is also necessary to maintain repression. Steroid receptor coactivator 2 (SRC2), which is known to play a vital role in GR-mediated repression of pro-inflammatory genes, was found to be dispensable for repression of glucocorticoid target genes sensitive to KDAC inhibition. At the promoters of these genes, KDAC inhibition did not result in altered nucleosome occupancy or histone H3 acetylation. Surprisingly, KDAC inhibition rapidly induced a significant decrease in H3K4Me2 at promoter nucleosomes with no corresponding change in H3K4Me3, suggesting the activation of the lysine demethylase, LSD1/KDM1A. Depletion of LSD1 expression via siRNA restored Dex-mediated repression in the presence of KDAC inhibitors, suggesting that LSD1 activation at these gene promoters is incompatible with transcriptional repression. Treatment with KDAC inhibitors does not alter cellular levels of LSD1 or its association with Dex-repressed gene promoters. Therefore, we conclude that Class I KDACs facilitate Dex-induced transcriptional repression by suppressing LSD1 complex activity at selected target gene promoters. Rather than facilitating repression of transcription, LSD1 opposes it in these gene contexts.
• Havas, A. P., Rodrigues, K. B., Demirjian, J. A., Tran, J., Scavello, M., Tula-Sanchez, A. A., Schmelz, M., & Smith, C. L. (2016). Belinostat and vincristine demonstrate mutually synergistic cytotoxicity associated with mitotic arrest and inhibition of polyploidy in a preclinical model of aggressive diffuse large B cell lymphoma. Cancer Biology and Therapy. doi:10.1080/15384047.2016.1250046
• Lake, A. D., Chaput, A. L., Novak, P., Cherrington, N. J., & Smith, C. L. (2016). Transcription Factor Binding Site Enrichment Analysis Predicts Drivers of Altered Gene Expression in Nonalcoholic Steatohepatitis. Biochemical Pharmacology, 122, 62-71. doi:10.1016/j.bcp.2016.11.006
• Dickinson, S. E., Rusche, J. J., Bec, S. L., Horn, D. J., Janda, J., Rim, S. H., Smith, C. L., & Bowden, G. T. (2015). The effect of sulforaphane on histone deacetylase activity in keratinocytes: Differences between in vitro and in vivo analyses. Molecular carcinogenesis, 54(11), 1513-20.
  More info

  Sulforaphane is a natural product found in broccoli, which is known to exert many different molecular effects in the cell, including inhibition of histone deacetylase (HDAC) enzymes. Here, we examine for the first time the potential for sulforaphane to inhibit HDACs in HaCaT keratinocytes and compare our results with those found using HCT116 colon cancer cells. Significant inhibition of HDAC activity in HCT116 nuclear extracts required prolonged exposure to sulforaphane in the presence of serum. Under the same conditions HaCaT nuclear extracts did not exhibit reduced HDAC activity with sulforaphane treatment. Both cell types displayed down-regulation of HDAC protein levels by sulforaphane treatment. Despite these reductions in HDAC family member protein levels, acetylation of marker proteins (acetylated Histone H3, H4, and tubulin) was decreased by sulforaphane treatment. Time-course analysis revealed that HDAC6, HDAC3, and acetylated histone H3 protein levels are significantly inhibited as early as 6 h into sulforaphane treatment. Transcript levels of HDAC6 are also suppressed after 48 h of treatment. These results suggest that HDAC activity noted in nuclear extracts is not always translated as expected to target protein acetylation patterns, despite dramatic inhibition of some HDAC protein levels. In addition, our data suggest that keratinocytes are at least partially resistant to the nuclear HDAC inhibitory effects of sulforaphane, which is exhibited in HCT116 and other cells.
• Kadiyala, V., & Smith, C. L. (2014). Minireview: The versatile roles of lysine deacetylases in steroid receptor signaling. Molecular endocrinology (Baltimore, Md.), 28(5), 607-21.
  More info

  Lysine deacetylases have been known to regulate nuclear receptor function for many years. In the unliganded state, nuclear receptors that form heterodimers with retinoid X receptors, such as the retinoic acid and thyroid hormone receptors, associate with deacetylases to repress target genes. In the case of steroid receptors, binding of an antagonist ligand was initially reported to induce association of deacetylases to prevent activation of target genes. Since then, deacetylases have been shown to have diverse functions in steroid receptor signaling, from regulating interactions with molecular chaperones to facilitating their ability to activate transcription. The purpose of this review is to summarize recent studies on the role of deacetylases in steroid receptor signaling, which show deacetylases to be highly versatile regulators of steroid receptor function.
• Kadiyala, V. -., Patrick, N., Mathieu, W., Jaime-Frias, R., Pookhao, N., An, L. -., & Smith, C. L. (2013). Class I lysine deacetylases facilitate glucocorticoid-induced transcription.. Journal of Biology Chemistry, 288, 28900-12.
• Kadiyala, V., Patrick, N. M., Mathieu, W., Jaime-Frias, R., Pookhao, N., An, L., & Smith, C. L. (2013). Class I lysine deacetylases facilitate glucocorticoid-induced transcription. The Journal of biological chemistry, 288(40), 28900-12.
  More info

  Nuclear receptors use lysine acetyltransferases and lysine deacetylases (KDACs) in regulating transcription through histone acetylation. Lysine acetyltransferases interact with steroid receptors upon binding of an agonist and are recruited to target genes. KDACs have been shown to interact with steroid receptors upon binding to an antagonist. We have shown previously that KDAC inhibitors (KDACis) potently repress the mouse mammary tumor virus promoter through transcriptional mechanisms and impair the ability of the glucocorticoid receptor (GR) to activate it, suggesting that KDACs can play a positive role in GR transactivation. In the current study, we extended this analysis to the entire GR transcriptome and found that the KDACi valproic acid impairs the ability of agonist-bound GR to activate about 50% of its target genes. This inhibition is largely due to impaired transcription rather than defective GR processing and was also observed using a structurally distinct KDACi. Depletion of KDAC1 expression mimicked the effects of KDACi in over half of the genes found to be impaired in GR transactivation. Simultaneous depletion of KDACs 1 and 2 caused full or partial impairment of several more GR target genes. Altogether we found that Class I KDAC activity facilitates GR-mediated activation at a sizable fraction of GR-activated target genes and that KDAC1 alone or in coordination with KDAC2 is required for efficient GR transactivation at many of these target genes. Finally, our work demonstrates that KDACi exposure has a significant impact on GR signaling and thus has ramifications for the clinical use of these drugs.
• Tula-Sanchez, A. A., Havas, A. P., Alonge, P. J., Klein, M. E., Doctor, S. R., Pinkston, W., Glinsmann-Gibson, B. J., Rimsza, L. M., & Smith, C. L. (2013). A model of sensitivity and resistance to histone deacetylase inhibitors in diffuse large B cell lymphoma: Role of cyclin-dependent kinase inhibitors. Cancer Biology and Therapy, 14(10), 949-961.
  More info

  PMID: 23982416;PMCID: PMC3926892;Abstract: Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma. While the initial treatment strategy is highly effective, relapse occurs in 40% of cases. Histone deacetylase inhibitors (HDAC i) are a promising class of anti-cancer drugs but their single agent efficacy against relapsed DLBCL has been variable, ranging from few complete/partial responses to some stable disease. However, most patients showed no response to HDAC i monotherapy for unknown reasons. Here we show that sensitivity and resistance to the hydroxamate HDAC i, PXD101, can be modeled in DLBCL cell lines. Sensitivity is characterized by G2/M arrest and apoptosis and resistance by reversible G1 growth arrest. These responses to PXD101 are independent of several negative prognostic indicators such as DLBCL subtype, BCL2 and MYC co-expression, and p53 mutation, suggesting that HDAC i might be used effectively against highly aggressive DLBCL tumors if they are combined with other therapeutics that overcome HDAC i resistance. Our investigation of mechanisms underlying HDAC i resistance showed that cyclin-dependent kinase inhibitors (CKIs), p21 and p27, are upregulated by PXD101 in a sustained fashion in resistant cell lines concomitant with decreased activity of the cyclin E/cdk2 complex and decreased Rb phosphorylation. PXD101 treatment results in increased association of CKI with the cyclin E/cdk2 complex in resistant cell lines but not in a sensitive line, indicating that the CKIs play a key role in G1 arrest. The results suggest several treatment strategies that might increase the efficacy of HDAC i against aggressive DLBCL. © 2013 Landes Bioscience.
• Smith, C. L., Lee, S. C., & Magklara, A. (2011). HDAC activity is required for efficient core promoter function at the mouse mammary tumor virus promoter. Journal of Biomedicine and Biotechnology, 2011.
  More info

  PMID: 21253530;PMCID: PMC3021843;Abstract: Histone deacetylases (HDACs) have been shown to be required for basal or inducible transcription at a variety of genes by poorly understood mechanisms. We demonstrated previously that HDAC inhibition rapidly repressed transcription from the mouse mammary tumor virus (MMTV) promoter by a mechanism that does not require the binding of upstream transcription factors. In the current study, we find that HDACs work through the core promoter sequences of MMTV as well as those of several cellular genes to facilitate transcriptional initiation through deacetylation of nonhistone proteins. © 2011 Sang C. Lee et al.
• Magklara, A., & Smith, C. L. (2009). A composite intronic element directs dynamic binding of the progesterone receptor and GATA-2. Molecular Endocrinology, 23(1), 61-73.
  More info

  PMID: 19036901;PMCID: PMC2646598;Abstract: The progesterone receptor (PR) plays a pivotal role in proper development and function of the mammary gland and has also been implicated in mammary tumorigenesis. PR is a ligand-activated transcription factor; however, relatively, little is known about its mechanisms of action at endogenous target promoters. The aim of our study was to identify a natural PR-responsive gene and investigate its transcriptional regulation in the mammary microenvironment. Our experiments revealed FKBP5 as a direct target of the PR, because it exhibited a rapid activation by progestin that was cycloheximide independent and correlated with recruitment of RNA polymerase II to the promoter. Site-directed mutagenesis and chromatin immunoprecipitation assays showed that progestin responsiveness is mediated through a composite element in the first intron, to which the PR binds concomitantly with GATA-2. Mutational analysis of the element revealed that the GATA-2 site is essential for progestin activation. Direct binding of PR to DNA contributes to the efficiency of activation but is not sufficient, suggesting that the receptor makes important protein-protein interactions as part of its mechanism of action at the FKBP5 promoter. Using chromatin immunoprecipitation assays we also determined that the intronic region is in communication with the promoter, probably via DNA looping. Time course analysis revealed a cyclical pattern of PR recruitment to the FKBP5 gene but a persistent recruitment to the mouse mammary tumor virus promoter, indicating that receptor cycling is a gene-specific phenomenon rather than a characteristic of the receptor itself. Our study offers new insight in the nature of PR-regulated transcription in mammary cancer cells. Copyright © 2009 by The Endocrine Society.
• Rodriguez-Collazo, P., Snyder, S. K., Chiffer, R. C., Bressler, E. A., Voss, T. C., Anderson, E. P., Genieser, H., & Smith, C. L. (2008). cAMP signaling regulates histone H3 phosphorylation and mitotic entry through a disruption of G2 progression. Experimental Cell Research, 314(15), 2855-2869.
  More info

  PMID: 18644368;Abstract: cAMP signaling is known to have significant effects on cell growth, either inhibitory or stimulatory depending on the cell type. Study of cAMP-induced growth inhibition in mammalian somatic cells has focused mainly on the combined role of protein kinase A (PKA) and mitogen-activated protein (MAP) kinases in regulation of progression through the G1 phase of the cell cycle. Here we show that cAMP signaling regulates histone H3 phosphorylation in a cell cycle-dependent fashion, increasing it in quiescent cells but dramatically reducing it in cycling cells. The latter is due to a rapid and dramatic loss of mitotic histone H3 phosphorylation caused by a disruption in G2 progression, as evidenced by the inhibition of mitotic entry and decreased activity of the CyclinB/Cdk1 kinase. The inhibition of G2 progression induced through cAMP signaling is dependent on expression of the catalytic subunit of PKA and is highly sensitive to intracellular cAMP concentration. The mechanism by which G2 progression is inhibited is independent of both DNA damage and MAP kinase signaling. Our results suggest that cAMP signaling activates a G2 checkpoint by a unique mechanism and provide new insight into normal cellular regulation of G2 progression. © 2008 Elsevier Inc.
• Rodriguez-Collazo, P., Snyder, S. K., Chiffer, R. C., Zlatanova, J., Leuba, S. H., & Smith, C. L. (2008). cAMP signaling induces rapid loss of histone H3 phosphorylation in mammary adenocarcinoma-derived cell lines. Experimental Cell Research, 314(1), 1-10.
  More info

  PMID: 17950276;Abstract: The phosphorylation of histone H3 is known to play a role in regulation of transcription as well as preparation of chromosomes for mitosis. Various signaling cascades induce H3 phosphorylation, particularly at genes activated by these pathways. In this study, we show that signaling can also have the opposite effect. Activators of cAMP signaling induce a rapid and potent loss of H3 phosphorylation. This effect is not mediated through a cAMP metabolite since a membrane-permeable form of AMP had no effect on H3 phosphorylation and a phosphodiesterase-resistant cAMP analog efficiently reduced it. cAMP is also the likely regulator of H3 phosphorylation under physiological conditions since only supra-pharmacological doses of cGMP induce the loss of H3 phosphorylation. The loss of phosphorylation is specific for histone H3 since we do not observe drastic losses in total phosphorylation of other histones. In addition, other H3 modifications are unaffected with the exception of lysine 9 methylation, which is elevated. Analysis of cell growth and cell cycle shows that cAMP signaling inhibits cell growth and arrests cells at both G1 and G2/M. Similar effects of cAMP signaling on H3 phosphorylation are observed in a variety of mammary adenocarcinoma-derived cell lines. In syngeneic human breast-derived cell lines, one diploid and non-transformed, the other derived from a ductal carcinoma, the loss of H3 phosphorylation is significantly more sensitive to cAMP concentration in the transformed cell line. © 2007 Elsevier Inc. All rights reserved.
• Smith, C. L. (2008). A shifting paradigm: Histone deacetylases and transcriptional activation. BioEssays, 30(1), 15-24.
  More info

  PMID: 18081007;Abstract: Transcriptional repression and silencing have been strongly associated with hypoacetylation of histones. Accordingly, histone deacetylases, which remove acetyl groups from histones, have been shown to participate in mechanisms of transcriptional repression. Therefore, current models of the role of acetylation in transcriptional regulation focus on the acetylation status of histones and designate histone acetyltransferases, which add acetyl groups to histones, as transcriptional coactivators and histone deacetylases as corepressors. In recent years, an accumulation of studies have shown that these enzymes also target non-histone proteins and that histone deacetylases have clear roles as coactivators at a variety of genes, some of which are key regulators of cell growth and survival. This review summarizes the evidence for histone deacetylases as coactivators and provides models of coactivation mechanisms, some of which integrate roles of acetylated histones and non-histone proteins in transcription. © 2007 Wiley Periodicals, Inc.
• Botos, J., Xian, W., Smith, D. F., & Smith, C. L. (2004). Progesterone Receptor Deficient in Chromatin Binding Has an Altered Cellular State. Journal of Biological Chemistry, 279(15), 15231-15239.
  More info

  PMID: 14744870;Abstract: Our previous work has shown that the progesterone receptor (PR) can exist in two distinct functional states in mammary adenocarcinoma cells. The differences in function included the ability to activate a promoter in organized chromatin, sensitivity to ligand, and ligand-independent activation. To determine whether these functional differences were because of altered cellular processing, we carried out biochemical analyses of the functionally distinct PRs. Although the majority of PR is localized to the nucleus, biochemical partitioning resulted in a loosely bound (cytosolic) fraction, and a tightly bound (nuclear) fraction. In the absence of progestins, the functionally distinct PRs differed significantly in partitioning between the two fractions. To characterize these fractions further, we analyzed interactions of unliganded PR with chaperones by coimmunoprecipitation. We determined that PR in the cytosolic fraction associated with hsp90 and p23. In contrast, PR in the nuclear fraction consisted of complexes containing hsp90, p23, and FKBP51 as well as PR that was dimerized and highly phosphorylated. Hormone treatment significantly reduced the formation of all PR-chaperone complexes. The hsp90 inhibitor, geldanamycin, similarly blocked transcriptional activity of both functionally distinct receptors. However, the two forms of the PR differed in their ability to associate with the mouse mammary tumor virus promoter in organized chromatin. These findings provide new information about the composition and distribution of mature progesterone receptor complexes in mammary adenocarcinoma cells, and suggest that differences in receptor subcellular distribution have a significant impact on their function. These findings also reveal that transiently expressed steroid receptors may not always be processed like their endogenous counterparts.
• Mulholland, N. M., Snyder, S. K., Kolla, S. S., & Smith, C. L. (2003). Chromatin-dependent regulation of the MMTV promoter by cAMP signaling is mediated through distinct pathways. Experimental Cell Research, 287(2), 361-373.
  More info

  PMID: 12837291;Abstract: The nucleoprotein structure of the mouse mammary tumor virus (MMTV) promoter defines its response to cAMP signaling. A stably replicating MMTV template in highly organized chromatin is repressed in the presence of cAMP, whereas a transiently transfected template with a disorganized structure is activated. In this study, we investigate the nature of the cAMP-induced signal(s) by which these opposing responses occur to gain insight into their mechanism. We demonstrate that the transcriptional changes observed at both templates are mediated through cAMP-dependent protein kinase A (PKA). In addition, the MMTV promoter lacks a consensus cAMP response element (CRE) and neither template requires cAMP response element-binding protein (CREB) to elicit a response to cAMP signaling. However, the responses of the two templates differ mechanistically in that the CREB-binding protein p300 potentiates activation from the transient template in a manner dependent on its Cys/His-rich region 3, but does not appear to affect the repression of the replicating chromatin template. Chromatin immunoprecipitation assays show that cAMP treatment results in a decrease in acetylation of histone H4, and in multiple modifications of histone H3 at specific nucleosomes in the promoter region of the stable MMTV template. These findings suggest novel CREB-independent, chromatin-dependent pathways for transcriptional regulation by cAMP. © 2003 Elsevier Science (USA). All rights reserved.
• Mulholland, N. M., Soeth, E., & Smith, C. L. (2003). Inhibition of MMTV transcription by HDAC inhibitors occurs independent of changes in chromatin remodeling and increased histone acetylation. Oncogene, 22(31), 4807-4818.
  More info

  PMID: 12894222;Abstract: Increased histone acetylation has been associated with activated gene transcription and decreased acetylation with repression. However, there is a growing number of genes known, which are downregulated by histone deacetylase (HDAC) inhibitors through unknown mechanisms. This study examines the mechanism by which the mouse mammary tumor virus (MMTV) promoter is repressed by the HDAC inhibitor, trichostatin A (TSA). We find that this repression is transcriptional in nature and that it occurs in the presence and absence of glucocorticoids. TSA decreases MMTV transcription at a rapid rate, reaching maximum in 30-60 min. In contrast with previous reports, the repression does not correlate with an inhibition of glucocorticoid-induced nuclease hypersensitivity or NF1-binding at the MMTV promoter. Surprisingly, TSA does not induce sizable increases in histone acetylation at the MMTV promoter nor does it inhibit histone deacetylation, which accompanies deactivation of the glucocorticoid-activated MMTV promoter. Repression of MMTV transcription by TSA does not depend on the chromatin organization of the promoter because a transiently transfected MMTV promoter construct with a disorganized nucleoprotein structure was also repressed by TSA treatment. Mutational analysis of the MMTV promoter indicates that repression by TSA is mediated through the TATA box region. These results suggest a novel mechanism that involves acetylation of nonhistone proteins necessary for basal transcription.
• Keeton, E. K., Fletcher, T. M., Baumann, C. T., Hager, G. L., & Smith, C. L. (2002). Glucocorticoid receptor domain requirements for chromatin remodeling and transcriptional activation of the mouse mammary tumor virus promoter in different nucleoprotein contexts. Journal of Biological Chemistry, 277(31), 28247-28255.
  More info

  PMID: 12029095;Abstract: The glucocorticoid receptor (GR) contains several activation domains, τ1 (AF-1), τ2, and AF-2, which were initially defined using transiently transfected reporter constructs. Using domain mutations in the context of full-length GR, this study defines those domains required for activation of the mouse mammary tumor virus (MMTV) promoter in two distinct nucleoprotein configurations. A transiently transfected MMTV template with a disorganized, accessible chromatin structure was largely dependent on the AF-2 domain for activation. In contrast, activation of an MMTV template in organized, replicated chromatin requires both domains but has a relatively larger dependence on the τ1 domain. Domain requirements for GR-induced chromatin remodeling of the latter template were also investigated. Mutation of the AF-2 helix 12 domain partially inhibits the induction of nuclease hypersensitivity, but the inhibition was relieved in the absence of τ1, suggesting the occurrence of an important interaction between the two domains. Further mutational analysis indicates that GR-induced chromatin remodeling requires the ligand-binding domain in the region of helix 3. Our study shows that the GR activation surfaces required for transcriptional modulation of a target promoter were determined in part by its chromatin structure. Within a particular cellular environment the GR appears to possess a significant degree of versatility in the mechanism by which it activates a target promoter.
• Soeth, E., Thurber, D. B., & Smith, C. L. (2002). The viral transactivator E1A regulates the mouse mammary tumor virus promoter in an isoform- and chromatin-specific manner. Journal of Biological Chemistry, 277(22), 19847-19854.
  More info

  PMID: 11909860;Abstract: Proteins encoded by the adenovirus E1A gene regulate both cellular and viral genes to mediate effects on cell cycle, differentiation, and cell growth control. We have identified the mouse mammary tumor virus (MMTV) promoter as a target of EIA action and investigated the role nucleoprotein structure plays in its response to E1A. Both 12 and 13 S forms target the MMTV promoter when it has a disorganized and accessible chromatin configuration. However, whereas the 13 S form is stimulatory, the 12 S form is repressive. When the MMTV promoter adopts an organized and repressed chromatin structure, it is targeted only by the 13 S form, which stimulates it. Although evidence indicates that EIA interacts with the SWI/SNF remodeling complex, E1A had no effect on chromatin remodeling at the MMTV promoter in organized chromatin. Analysis of EIA mutants showed that stimulation of the MMTV promoter is mediated solely through conserved region 3 and does not require interaction with Rb, p300/CBP associated factor, or CBP/p300. Imaging analysis showed that EIA colocalizes with MMTV sequences in vivo, suggesting that it functions directly at the promoter. These results indicate that EIA stimulates the MMTV promoter in a fashion independent of chromatin conformation and through a direct mechanism involving interaction with the basal transcription machinery.
• Giannoukos, G., Szapary, D., Smith, C. L., E., J., & Simons Jr., S. S. (2001). New antiprogestins with partial agonist activity: Potential selective progesterone receptor modulators (SPRMs) and probes for receptor- and coregulator-induced changes in progesterone receptor induction properties. Molecular Endocrinology, 15(2), 255-270.
  More info

  PMID: 11158332;Abstract: A pharmacologically relevant property of steroid hormone-regulated gene induction is the partial agonist activity of antisteroid complexes. We now report that dexamethasone-mesylate (Dex-Mes) and dexamethasone-oxetanone (Dex-Ox), each a derivative of the glucocorticoid-selective steroid dexamethasone (Dex), are two new antiprogestins with significant amounts of agonist activity with both the A and B isoforms of progesterone receptor (PR), for different progesterone-responsive elements, and in several cell lines. These compounds continue to display activity under conditions where another partial antiprogestin (RTI-020) is inactive. These new antiprogestins were used to determine whether the partial agonist activity of PR complexes can be modified by changing concentrations of receptor or coregulator, as we have recently demonstrated for glucocorticoid receptors (GRs). Because GR and coregulator concentrations simultaneously altered the position of the physiologically relevant dose-response curve, and associated EC50, of GR-agonist complexes, we also examined this phenomenon with PR. We find that elevated PR or transcriptional intermediary factor 2 (TIF2) concentrations increase the partial agonist activity of Dex-Mes and Dex-Ox, and the EC50 of agonists, independently of changes in total gene transactivation. Furthermore, the corepressors SMRT (silencing mediator for retinoid and thyroid receptors) and NCoR (nuclear receptor corepressor) each suppresses gene induction but NCoR acts opposite to SMRT and, like the coactivator TIF2, reduces the EC50 and increases the partial agonist activity of antiprogestins. These comparable responses of GR and PR suggest that variations in receptor and coregulator concentrations may be a general mechanism for altering the induction properties of other steroid receptors. Finally, the magnitude of coregulator effects on PR induction properties are often not identical for agonists and the new antagonists, suggesting subtle mechanistic differences. These properties of Dex-Mes and Dex-Ox, plus the sensitivity of their activity to cellular differences in PR and coregulator concentrations, make these steroids potential new SPRMs (selective progesterone receptor modulators) that should prove useful as probes of PR induction properties.
• Sheldon, L. A., Becker, M., & Smith, C. L. (2001). Steroid Hormone Receptor-mediated Histone Deacetylation and Transcription at the Mouse Mammary Tumor Virus Promoter. Journal of Biological Chemistry, 276(35), 32423-32426.
  More info

  PMID: 11448945;Abstract: Acetylation of lysines in histones H3 and H4 N-terminal tails is associated with transcriptional activation and deacetylation with repression. Our studies with the mouse mammary tumor virus (MMTV) promoter in chromatin show significant levels of acetylation at promoter proximal and distal regions prior to transactivation. Upon activation with glucocorticoids or progestins, promoter proximal histones become deacetylated within the region of inducible nuclease hypersensitivity. The deacetylation lags behind the initiation of transcription, indicating a role in post-activation regulation. Our results indicate a novel mechanism by which target promoters are regulated by steroid receptors and chromatin modification machinery.
• List, H., Smith, C. L., Martinez, E., Harris, V. K., Danielsen, M., & Riegel, A. T. (2000). Effects of antiandrogens on chromatin remodeling and transcription of the integrated mouse mammary tumor virus promoter. Experimental Cell Research, 260(1), 160-165.
  More info

  PMID: 11010820;Abstract: Inhibition of the ligand-activated androgen receptor (AR) by antiandrogens plays an important role in the treatment of various hyperandrogenic disorders including prostate cancer. However, the molecular mechanisms of antiandrogen activity in vivo remain unclear. In this study we analyzed the effects of cyproterone acetate (CPA), flutamide (F), and hydroxyflutamide (OHF) on transcriptional activation and chromatin remodeling of the genomically integrated mouse mammary tumor virus (MMTV) promoter. This promoter has provided an excellent model system to study the impact of steroid hormones on transcriptional activation in the context of a defined chromatin structure. The MMTV hormone response element is positioned on a phased nucleosome, which becomes remodeled in response to steroids. We utilized this model system in mouse L-cell fibroblasts that contain a stably integrated MMTV promoter. In these cells, dihydrotestosterone (DHT) induced a large increase of AR protein levels that correlated with transcriptional activation and chromatin remodeling of the MMTV promoter. Coadministration of DHT and CPA or DHT and OHF in these cells inhibited the increase of AR levels, which resulted in a strong blockage of transcriptional activation and chromatin remodeling of the MMTV promoter. In contrast, F had no significant influence on these activities. We conclude that a major portion of the antiandrogenic effects of CPA and OHF in vivo are mediated by the reduction of AR levels. (C) 2000 Academic Press.
• Smith, C. L., Wolford, R. G., O'Neill, T. B., & Hager, G. L. (2000). Characterization of transiently and constitutively expressed progesterone receptors: Evidence for two functional states. Molecular Endocrinology, 14(7), 956-971.
  More info

  PMID: 10894147;Abstract: Activated steroid receptors induce chromatin remodeling events in the promoters of some target genes. We previously reported that transiently expressed progesterone receptor (PR) cannot activate mouse mammary tumor virus (MMTV) promoter when it adopts the form of ordered chromatin. However, when expressed continuously, the PR acquires this ability. In this study we explored whether this gain of function occurs through alterations in nucleoprotein structure at the MMTV promoter or through changes in receptor status. We observed no major structural differences at the MMTV promoter in the presence of constitutively expressed PR and found its mechanism of activation to be very similar to that of the glucocorticoid receptor (GR). However, a systematic comparison of the functional behavior of the transiently and constitutively expressed PR elucidated significant differences. The transiently expressed PR is activated in the absence of ligand by cAMP and by components in FBS and has significantly increased sensitivity to progestins. In contrast, the constitutively expressed PR is refractory to activation by cAMP and serum and has normal sensitivity to its ligand. In addition, while the PR is localized to the nucleus in both cases, a significant fraction of the transiently expressed PR is tightly bound to the nucleus even in the absence of ligand, while the majority of constitutively expressed PR is not. These results strongly suggest that the PR undergoes processing in the cell subsequent to its initial expression and that this processing is important for various aspects of its function, including its ability to productively interact with target genes that require chromatin remodeling for activation.
• Lim, C. S., Baumann, C. T., Htun, H., Xian, W., Irie, M., Smith, C. L., & Hager, G. L. (1999). Differential localization and activity of the A- and B-forms of the human progesterone receptor using green fluorescent protein chimeras. Molecular Endocrinology, 13(3), 366-375.
  More info

  PMID: 10076994;Abstract: Subcellular localization and transcriptional activity of green fluorescent protein-progesterone receptor A and B chimeras (GFP-PRA and GFP- PRB) were examined in living mammalian cells. Both GFP-PRA and B chimeras were found to be similar in transcriptional activity compared with their non- GFP counterparts. GFP-PRA and PRA were both weakly active, while GFP-PRB and PRB gave a 20- to 40-fold induction using a reporter gene containing the full-length mouse mammary tumor virus long-terminal repeat linked to the luciferase gene (pLTRluc). Using fluorescence microscopy, nuclear/cytoplasmic distributions for the unliganded and hormone activated forms of GFP-PRA and GFP-PRB were characterized. The two forms of the receptor were found to have distinct intracellular distributions; GFP-PRA was found to be more nuclear than GFP-PRB in four cell lines examined. The causes for and implications of this differential localization of the A and B forms of the human PR are discussed.
• List, H., Smith, C. L., Rodriguez, O., Danielsen, M., & Riegel, A. T. (1999). Inhibition of histone deacetylation augments dihydrotestosterone induction of androgen receptor levels: An explanation for trichostatin a effects on androgen-induced chromatin remodeling and transcription of the mouse mammary tumor virus promoter. Experimental Cell Research, 252(2), 471-478.
  More info

  PMID: 10527637;Abstract: The integrated mouse mammary tumor virus (MMTV) promoter has provided an excellent model system with which to study the impact of steroid hormones on transcriptional activation in the context of a defined chromatin structure. The hormone response element (HRE) of this promoter is positioned on a phased nucleosome which becomes remodeled in response to steroids. One possible mechanism of chromatin remodeling by steroid receptors could involve recruitment of coactivators which alter the histone acetylation status of the HRE nucleosome. To examine how the androgen receptor (AR) influences transcription and chromatin remodeling and to assess whether changes in histone acetylation are involved in these effects, we determined whether the specific histone deacetylase inhibitor trichostatin A (TSA) influenced basal- and androgen-mediated transcriptional activation of the integrated MMTV promoter in the mouse L-cell fibroblast cell line 29+. These cells harbor the MMTV promoter integrated in the genome and express only one steroid hormone receptor subtype, i.e., the AR. Surprisingly, we found that treatment of the cells with TSA alone had virtually no effect on transcription and chromatin remodeling of the MMTV promoter nor on AR levels. However, pretreatment with TSA augmented the DHT effects on all three parameters. These results suggest that histone acetylation changes at the MMTV B nucleosome per se are not alone sufficient to induce chromatin remodeling and subsequent induction of MMTV transcription. Rather, the histone deacetylase inhibitor TSA exerts a portion of its effect on MMTV chromatin remodeling and transcriptional activation indirectly through increases in AR levels.
• Sheldon, L. A., Smith, C. L., Bodwell, J. E., Munck, A. U., & Hager, G. L. (1999). A ligand binding domain mutation in the mouse glucocorticoid receptor functionally links chromatin remodeling and transcription initiation. Molecular and Cellular Biology, 19(12), 8146-8157.
  More info

  PMID: 10567540;PMCID: PMC84899;Abstract: We utilized the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) in vivo to understand how the interaction of the glucocorticoid receptor (GR) with a nucleosome-assembled promoter allows access of factors required for the transition from a repressed promoter to a derepressed, transcriptionally competent promoter. A mutation (C644G) in the ligand binding domain (LBD) of the mouse GR has provided information regarding the steps required in the derepression/activation process and in the functional significance of the two major transcriptional activation domains, AF1 and AF2. The mutant GR activates transcription from a transiently transfected promoter that has a disordered nucleosomal structure, though significantly less well than the wild-type GR. With an integrated, replicated promoter, which is assembled in an ordered nucleosomal array, the mutant GR does not activate transcription, and it fails to induce chromatin remodeling of the MMTV LTR promoter, as indicated by nuclease accessibility assays. Together, these findings support a two-step model for the transition of a nucleosome- assembled, repressed promoter to its transcriptionally active, derepressed form. In addition, we find that the C-terminal GR mutation is dominant over the transcription activation function of the N-terminal GR activation domain. These findings suggest that the primary activation function of the C-terminal activation domain is different from the function of the N-terminal activation domain and that it is required for derepression of the chromatin-repressed MMTV promoter.
• Hager, G. L., Smith, C. L., Fragoso, G., Wolford, R., Walker, D., Barsony, J., & Htun, H. (1998). Intranuclear trafficking and gene targeting by members of the steroid/nuclear receptor superfamily. Journal of Steroid Biochemistry and Molecular Biology, 65(1-6), 125-132.
  More info

  PMID: 9699865;Abstract: Upon binding to regulatory elements in mammalian chromosomes, steroid receptors induce specific transitions in the nucleoprotein structure of the template. These transitions reflect, in part, the reorganization of chromatin structure to permit interaction of secondary factors with target sequences in promoter regulatory regions. Steroid receptors represent a class of transcriptional activators that are able to interact with repressed nucleoprotein templates and recruit necessary activities for chromatin remodeling. The ligand-induced movement of nuclear receptors from inactive states, either in the cytoplasm or in the nucleus, to productive interactions with chromatin is complex and likely reflects the interaction with multiple protein complexes and subcellular structures. Regulation of gene expression by nuclear receptors is thus mediated through the subcellular distribution of inactive receptors, the redistribution of activated receptor complexes to appropriate nuclear domains, the reorganization of chromatin structures for interaction with soluble components of the nucleoplasm, and direct protein- protein contacts between receptors and the basal transcription apparatus.
• Smith, C. L., & Hager, G. L. (1997). Transcriptional regulation of mammalian genes in vivo. A tale of two templates. Journal of Biological Chemistry, 272(44), 27493-27496.
  More info

  PMID: 9346875;Abstract: The study of mammalian gene expression through the use of transient transfection assays has greatly expanded our knowledge of transcriptional mechanisms. However, transfected promotor constructs do not always serve as appropriate 'stand-ins' for endogenous genes, particularly in cases where chromatin remodeling must take place. The examples described above indicate that a level of caution is advised when studying regulation of various promoters and transcription factor function with the use of transient transfection assays. When possible, function of the corresponding endogenous promoters should be tested to assess the validity of regulatory mechanisms defined on transiently transfected, non-replicating templates. Future studies using template comparison as a tool will undoubtedly identify which transcription factors are critical in initiating essential changes in chromatin structure at endogenous target genes and assist in the elucidation of mechanisms involved in chromatin transitions. As in the MyoD experiments (36), these studies will also allow identification of functional domains in these factors, which are necessary for chromatin remodeling. Functional differences between transiently transfected and stable replicating templates need not be considered artifactual but rather can be exploited to identify and characterize regulatory mechanisms that involve chromatin components. Full understanding of gene expression in vivo will not be achieved until these mechanisms are understood in detail.
• Smith, C. L., Htun, H., Wolford, R. G., & Hager, G. L. (1997). Differential activity of progesterone and glucocorticoid receptors on mouse mammary tumor virus templates differing in chromatin structure. Journal of Biological Chemistry, 272(22), 14227-14235.
  More info

  PMID: 9162055;Abstract: In vivo, transcription factors interact with promoters having complex nucleoprotein structures. The transiently expressed progesterone receptor (PR) efficiently activates a transfected mouse mammary tumor virus (MMTV) promoter but is a poor activator of the MMTV promoter when it acquires an ordered chromatin structure as an endogenous, replicating gene. We show that the deficiency in PR activity is not due to insufficient expression of either B or A isoforms or competition between the two types of MMTV templates. Rather, this deficiency reflects an inability to induce the chromatin remodeling event that is required for activation of the replicated MMTV template. To determine whether this characteristic is common to transiently expressed steroid receptors or specific to the PR, we examined the activity of transiently expressed glucocorticoid (GR) receptor. Unlike the PR, the transiently expressed GR is an effective activator of both MMTV templates and efficiently induces the necessary chromatin remodeling event at the replicated template. These results indicate that the GR and PR have unique requirements for activation of promoters with ordered chromatin structure. These differences may provide a mechanism for establishing target gene specificity in vivo for steroid receptors that recognize and bind to identical DNA sequences.
• Barsony, J., Renyi, I., McKoy, W., Kang, H. C., Haugland, R. P., & Smith, C. L. (1995). Development of a biologically active fluorescent-labeled calcitriol and its use to study hormone binding to the vitamin D receptor. Analytical Biochemistry, 229(1), 68-79.
  More info

  PMID: 8533897;Abstract: To gain better insight into the mechanism of steroid receptor activation and calcitriol action, we have developed the first pharmacologically relevant fluorescent-labeled ligand for the vitamin D receptor (VDR). Purity and structure of three BODIPY-labeled calcitriol derivatives were characterized by TLC, HPLC, and 1H-NMR spectroscopy. 3β-BODIPY-calcitriol was the most potent derivative to induce 25-hydroxyvitamin D3 24-hydroxylase activity and to inhibit cell proliferation. It was taken up rapidly and specifically and was not cleaved by endogenous esterases. 3β-BODIPY-calcitriol also retained high-affinity binding to the VDR. Hormone binding to the receptor was measured by spectrofluorometry in high-salt extracts from cultured cells with wild-type VDR, from cells virally over-expressing the human VDR, and in intact cells with and without VDR. Results from fluorescent binding studies agreed with results from radioligand assays. The most useful feature of this reagent is that its fluorescence emission increases severalfold upon binding to VDR. This allows direct monitoring by microscopy of ligand receptor interactions in living cells. Fluorescent-labeled calcitriol can be a valuable diagnostic tool for cancer research and is essential for exploring the subcellular localization of VDRs.
• Nobukuni, Y., Smith, C. L., Hager, G. L., & Detera-Wadleigh, S. D. (1995). Characterization of the human glucocorticoid receptor promoter. Biochemistry, 34(25), 8207-8214.
  More info

  PMID: 7794935;Abstract: To elucidate the functional elements that are involved in the regulation of the human glucocorticoid receptor (hGR) gene, transient expression, DNase I footprinting, and gel mobility shift analyses were conducted. We found that the hGR promoter region between -700 and +38 bp contained 11 footprinted sites. Deletion of the -374 to -183 bp region, which is highly conserved between human and mouse (93%), induced a 5-24-fold reduction in promoter activity in HeLa, NIH3T3, CV1, and HepG2 cells. Three footprints, FP5, FP6, and FP7, were shown to map to this region. In particular, the FP7 site was found to be within the -374 to -347 bp region. Deletion of this region triggered a significant decline in promoter activity in HeLa and NIH3T3 cells but not in HepG2 cells. AP2 was found to bind FP7. In HepG2 cells AP2 elicited transactivation of the hGR promoter activity. Transfection data revealed that the upstream GC box-rich fragment between -700 and -375 bp induced a 4-7-fold activation of the heterologous tk promoter in an orientation-independent manner. Our studies demonstrate that several transcription factors are involved in regulating GR expression and that AP2 could function as an important positive regulator of GR promoter activity.
• Pennie, W. D., Hager, G. L., & Smith, C. L. (1995). Nucleoprotein structure influences the response of the mouse mammary tumor virus promoter to activation of the cyclic AMP signalling pathway. Molecular and Cellular Biology, 15(4), 2125-2134.
  More info

  PMID: 7891707;PMCID: PMC230440;Abstract: Recent studies have provided evidence of crosstalk between steroid receptors and cyclic AMP (cAMP) signalling pathways in the regulation of gene expression. A synergism between intracellular phosphorylation inducers and either glucocorticoids or progestins has been shown to occur during activation of the mouse mammary tumor virus (MMTV) promoter. We have investigated the effect of 8-Br-cAMP and okadaic acid, modulators of cellular kinases and phosphatases, on the hormone-induced activation of the MMTV promoter in two forms: a transiently transfected template with a disorganized, accessible nucleoprotein structure and a stably replicating template with an ordered, inaccessible nucleoprotein structure. Both okadaic acid and 8-Br-cAMP synergize significantly with either glucocorticoids or progestins in activating the transiently transfected MMTV template. In contrast, 8-Br-cAMP, but not okadaic acid, is antagonistic to hormone- induced activation of the stably replicating MMTV template. Nuclear run-on experiments demonstrate that this inhibition is a transcriptional effect on both hormone-induced transcription and basal transcription. Surprisingly, 8- Br-cAMP does not inhibit glucocorticoid-induced changes in restriction enzyme access and nuclear factor 1 binding. However, association of a complex with the TATA box region is inhibited in the presence of 8-Br-cAMP. Thus, cAMP treatment interferes with the initiation process but does not inhibit interaction of the receptor with the template. Since the replicated, ordered MMTV templates and the transfected, disorganized templates show opposite responses to 8-Br-cAMP treatment, we conclude that chromatin structure can influence the response of a promoter to activation of the cAMP signalling pathway.
• Pavlin, D., Bedalov, A., Kronenberg, M. S., Kream, B. E., Rowe, D. W., Smith, C. L., Pike, J. W., & Lichtler, A. C. (1994). Analysis of regulatory regions in the COL1A1 gene responsible for 1,25-Dihydroxyvitamin D₃-mediated transcriptional repression in osteoblastic cells. Journal of Cellular Biochemistry, 56(4), 490-501.
  More info

  PMID: 7890807;Abstract: The synthesis of type I collagen in bone cells is inhibited by the calcium-regulating hormone 1,25-dihydroxyvitamin D3. Earlier work from our laboratories has indicated that vitamin D regulation is at the level of transcription, based on results from both nuclear run-off assays and functional promoter analysis of a hybrid gene consisting of a 3.6 kb COL1A1 promoter fragment fused to the chloramphenicol acetyltransferase reporter gene. In the present study, we investigated the molecular basis for vitamin D-mediated transcriptional repression of the COL1A1 gene and report the identification of a region within the COL1A1 upstream promoter (the HindIII-PstI restriction fragment between nucleotides -2295 and -1670) which is necessary for 1,25-dihydroxyvitamin D3 responsiveness in osteoblastic cells. This hormone-mediated inhibitory effect on the marker gene parallels the inhibition of the endogenous collagen gene. A 41 bp fragment from this region (between nucleotides -2256 and -2216) contains a sequence which is very similar to vitamin D-responsive elements identified in the osteocalcin gene. Extracts from cultured cells which express a high level of vitamin D receptor contain a hormone:receptor complex that binds specifically to this 41 bp fragment, as demonstrated by bandshift analysis. However, deletion of this vitamin D receptor binding region from either a -3.5 kb or a -2.3 kb promoter fragment did not abolish vitamin D responsiveness, These results indicate that a vitamin D response element similar to that described for other vitamin D responsive genes (osteocalcin and osteopontin) does not alone mediate the repression of COL1A1 by 1,25-dihydroxyvitamin D3.
• Smith, C. L., Archer, T. K., Hamlin-Green, G., & Hager, G. L. (1993). Newly expressed progesterone receptor cannot activate stable, replicated mouse mammary tumor virus templates but acquires transactivation potential upon continuous expression. Proceedings of the National Academy of Sciences of the United States of America, 90(23), 11202-11206.
  More info

  PMID: 8248228;PMCID: PMC47950;Abstract: During development and differentiation, the expression of transcription factors is regulated in a temporal fashion. Newly expressed transcription factors must interact productively with target genes organized in chromatin. Although the mechanisms governing factor binding to chromatin templates are not well understood, it is now clear that template access can be dramatically influenced by nucleoprotein structure. We have examined the ability of a well characterized transactivator, the progesterone receptor (PR), to activate the mouse mammary tumor virus (MMTV) promoter organized either in stable, replicating templates that have a highly ordered nucleosome structure or as transiently transfected DNA, which adopts a less-defined structure. If the PR is transiently expressed in cells harboring both replicated and transient MMTV reporter constructs, it cannot significantly activate the stable replicated MMTV template. In contrast, when PR cDNA is stably inserted into the same cells and constitutively expressed, it gains the ability to activate both chromosomal and transiently introduced templates. These results demonstrate that newly expressed PR is not competent to activate the MMTV template in its native nucleoprotein conformation but acquires this ability upon prolonged expression in replicating cells.
• Smith, C. L., Hager, G. L., Pike, J. W., & Marx, S. J. (1991). Overexpression of the human vitamin D₃ receptor in mammalian cells using recombinant adenovirus vectors. Molecular Endocrinology, 5(6), 867-878.
  More info

  PMID: 1656244;Abstract: The human vitamin D3 receptor (hVDR) cDNA was cloned into the E1 region of the adenovirus genome to generate recombinant viruses which were used to infect 293 (adenovirus-transformed human fetal kidney) cells. High salt extracts from cells infected with the recombinant viruses were subjected to immunoblot analysis using a monoclonal antibody to chicken VDR and were shown to contain large quantities of a protein of approximately 50 kDa with a migration identical to that of the hVDR in T47D (human mammary adenocarcinoma) cells. Scatchard analysis showed that the infected cells express approximately 100-fold more receptor than T47D cells and that this receptor binds 1,25-dihydroxyvitamin D3 with high affinity. The overexpressed hVDR also binds to DNA-cellulose and is eluted with a KCI concentration similar to that determined for fully active endogenous VDR. Nuclear extracts from cells infected with the hVDR-expressing adenoviruses contain an activity that specifically binds an oligonucleotide with sequences from the rat osteocalcin vitamin D3 response element, as determined by gel mobility shift. This interaction can be inhibited by the presence of an anti-VDR antibody, but not by nonspecific immunoglobulins. We conclude, there-fore, that the overexpressed receptor has the ligand-and DNA-binding characteristics defined for endogenous VDR and that adenoviruses can be used to efficiently express large quantities of functional hVDR in a human cell line. Finally, a second binding activity, specific for the vitamin D response element, but distinct from the VDR, has been identified in extracts from uninfected cells.
• Barsony, J., McKoy, W., Smith, C., Marco, L. D., Kohidai, L., Liberman, U. A., & Marx, S. J. (1990). Heterogeneous coupling mechanisms between vitamin D receptors and their target actions. Calcium regulation and bone metabolism. Basic and clinical aspects: proceedings of the 10th International Conference on calcium regulating hormones and bone metabolism. ICS886, 174-178.
• Smith, C. L., Nordloh, P. W., & Chiu, J. -. (1989). The role of methylation in regulating the expression of the alpha-fetoprotein gene in developing rat liver and hepatoma cell lines. Molecular Carcinogenesis, 2(5), 287-297.
  More info

  PMID: 2481456;Abstract: We have examined four possible sites of methylation in the 5' flanking region of the alpha-fetoprotein (AFP) gene during liver development in the rat, paying particular attention to the neonatal period, in which AFP gene transcription changes rapidly. These sites are found in Mspl/Hpall sites located at -4197, -3038, -2431, and +3 bp relative to the transcription start site. Three of these sites are associated with sequence regions important for the regulation of AFP gene transcription. We found that, in general, the 5' flanking region of the gene was methylated more in the adult liver than in the livers of fetal and neonatal rats. In addition, the degree of methylation of all four sites examined was increased in the adult liver. One of these sites showed increased methylation as AFP gene activity decreased, whereas the others became more methylated only after transcriptional activity of the gene had ceased. In particular, the site (+3 bp) just adjacent to the transcriptional initiation site of the gene was fully methylated in the adult liver. In various rat hepatoma and liver cell lines methylation of this same site showed a particularly close correlation with the amount of transcriptional activity of the AFP promoter in these cell lines. Treatment of the hepatoma and liver cell lines with dexamethasone, which influences AFP gene expression, did not result in any changes in methylation of these sites in the 5' flanking region.
• Checovich, W. J., Fitch, W. L., Krauss, R. M., Smith, M. P., Rapacz, J., Smith, C. L., & Attie, A. D. (1988). Defective catabolism and abnormal composition of low-density lipoproteins from mutant pigs with hypercholesterolemia. Biochemistry, 27(6), 1934-1941.
  More info

  PMID: 3378039;Abstract: Metabolic and chemical properties of low-density lipoproteins (LDLs) were studied in a strain of pigs carrying a specific apo-B allele associated with hypercholesterolemia and premature atherosclerosis. LDL mass was significantly greater in mutant than in control pigs (400 ±55 mg/dL vs 103 ±26 mg/dL), as was LDL cholesterol. When normal and mutant LDLs were injected into the bloodstream of normal pigs, the fractional catabolic rate (FCR) of mutant LDL was about 30% lower than that of control LDL. In mutant pigs, the mean FCRs of mutant and control LDL were similar, although they were much lower than the corresponding FCRs observed in normal pigs. The density profile of LDL particles differed in control and mutant pigs; the peak LDL flotation rate was shifted from Sf0 = 5.3 ±1.9 in controls to a more buoyant 7.4 ±0.5 in mutants. The elevation of LDL in the mutants was restricted to the most buoyant LDL subspecies. This subpopulation of mutant LDL was enriched with cholesteryl ester (47% vs 37%) and depleted of triglyceride, relative to LDL of similar density and size in controls. The lipid compositions of the denser LDL subpopulations (ρ> l .043 g/mL) were similar in mutants and controls. We conclude that the hypercholesterolemia of these mutant pigs is accounted for by defective catabolism of LDL. The buoyant cholesterol ester enriched LDL subspecies that accumulate in plasma may contribute to the accelerated atherogenesis that occurs in these animals. © 1988 American Chemical Society.
• Sakuma, K., Cook, J. R., Smith, C. L., & Chiu, J. (1987). Methylation of the alpha-fetoprotein gene in isogenic rat hepatoma and liver cell lines. Biochemical and Biophysical Research Communications, 143(2), 447-453.
  More info

  PMID: 2436613;Abstract: We have found that DNA methylation is inversely correlated with alpha-fetoprotein (AFP) gene expression in a series of isogenic rat hepatoma cell lines. The 5′ end of the gene is extensively demethylated in AFP-producing cells and is highly methylated in cell lines which do not produce AFP. Glucocorticoid affects markedly the synthesis of AFP in the hepatoma cells. However, methylation patterns of cell lines which were treated with dexamethasone were not different from those of control cells, indicating that glucocorticoid action on AFP gene expression does not alter DNA methylation in this region of the gene. © 1987.
• Cote, G., Wang, Z., Smith, C., & Chiu, J. F. (1985). Alpha-fetoprotein gene binding proteins. Proceedings of the American Association for Cancer Research, VOL. 26, No. 6.

Presentations

• Smith, C. L. (2017, October). Repressors turned Activators: Understanding how histone deacetylases facilitate(!) transcription. Biological Chemistry Program Journal Club. University of Arizona: Biological Chemistry Program.
• Smith, C. L. (2015, February). Developing Effective Strategies for the Use of Histone Deacetylase Inhibitors in Treatment of Lymphoma. Clinical Collaborative Grand Rounds. University of Arizona Cancer Center: University of Arizona Cancer Center.
• Smith, C. L. (2015, July). Lysine Deacetylases and Lysine Demethylases in GR-Regulated Transcription. Transcription Factor and Chromatin Biology Interest Groups Joint Seminar. National Institutes of Health: National Cancer Institute/Cancer for Cancer REsearch.

Poster Presentations

• Smith, C. L. (2019, August). Inhibition of HDAC3 is Crucial for Achieving a Cytotoxic Response to Pan-HDACi in Diffuse Large B Cell Lymphoma. FASEB Conference on Reversible Acetylation of Proteins in Health and Disease. Lisbon, Protugal: FASEB.
• Smith, C. L. (2017, August). Class I KDACs that facilitate glucocorticoid-induced gene activation through multiple mechanisms.. Reversible Acetylation in Health & Disease (HDACs). Big Sky, Montana: FASEB Research Conferences.
• Smith, C. L. (2017, August). Dissecting HDAC-specific roles in growth and survival of diffuse large B cell lymphoma cells: Regulation of Cell Cycle, Apoptosis, and Polyploidy. Reversible Acetylation in Health & Disease (HDACs). Big Sky, Montana: FASEB Research Conferences.