Resume

• 1991

  English

  Doctor of Philosophy (Ph.D.)

  Molecular & Cellular Biology

  Penn State University

• 1987

  Bachelor's Degree

  Biotechnology

  Phi Delta Theta fraternity

  Rochester Institute of Technology

• Student Development

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  POU1F1-mediated activation of hGH-N by deoxyribonuclease hypersensitive site II of the human growth hormone locus control region

  Andrew Franklin

  J. Kaitlin Morrison

  Holly Jefferson

  Tamra Hunsaker

  Journal of Molecular Biology

  The human growth hormone gene (hGH-N) is regulated by a distal locus control region (LCR) composed of five deoxyribonuclease I hypersensitive sites (HSs). The region encompassing HSI and HSII contains the predominant pituitary somatotrope-specific hGH-N activation function of the LCR. This activity was attributed primarily to POU1F1 (Pit-1) elements at HSI

  as linkage to HSI was sufficient for properly regulated hGH-N expression in transgenic mice

  while HSII alone had no activity. However

  the presence of HSII in conjunction with HSI further enhanced hGH-N transgene expression

  indicating additional determinants of pituitary hGH-N activation in the HSII region

  but limitations of transgenic models and previous ex vivo systems have prevented the characterization of HSII. In the present study

  we employ a novel minichromosome model of the hGH-N regulatory domain and show that HSII confers robust POU1F1-dependent activation of hGH-N in this system. This effect was accompanied by POU1F1-dependent histone acetylation and methylation throughout the minichromosome LCR/hGH-N domain. A series of in vitro DNA binding experiments revealed that POU1F1 binds to multiple sites at HSII

  consistent with a direct role in HSII function. Remarkably

  POU1F1 binding was localized in part to the 3' untranslated region of a primate-specific LINE-1 (long interspersed nuclear element 1) retrotransposon

  suggesting that its insertion during primate evolution may have conferred function to the HSII region in the context of pituitary GH gene regulation. These observations clarify the function of HSII

  expanding the role of POU1F1 in hGH LCR activity

  and provide insight on the molecular evolution of the LCR.

  POU1F1-mediated activation of hGH-N by deoxyribonuclease hypersensitive site II of the human growth hormone locus control region

  Perrine Lallemand

  Jenifer Fenton

  EPA and DHA are not biologically equivalent; however

  their individual activity on B cells is unknown. We previously reported fish oil enhanced murine B-cell activity in obesity. To distinguish between the effects of EPA and DHA

  we studied the ethyl esters of EPA and DHA on murine B-cell function as a function of time. We first demonstrate that EPA and DHA maintained the obese phenotype

  with no improvements in fat mass

  adipose inflammatory cytokines

  fasting insulin

  or glucose clearance. We then tested the hypothesis that EPA and DHA would increase the frequency of splenic B cells. EPA and DHA differentially enhanced the frequency and/or percentage of select B-cell subsets

  correlating with increased natural serum IgM and cecal IgA. We next determined the activities of EPA and DHA on ex vivo production of cytokines upon lipopolysaccharide stimulation of B cells. EPA and DHA

  in a time-dependent manner

  enhanced B-cell cytokines with DHA notably increasing IL-10. At the molecular level

  EPA and DHA differentially enhanced the formation of ordered microdomains but had no effect on Toll-like receptor 4 mobility. Overall

  the results establish differential effects of EPA and DHA in a time-dependent manner on B-cell activity in obesity

  which has implications for future clinical studies.

  Eicosapentaenoic and docosahexaenoic acid ethyl esters differentially enhance B-cell activity in murine obesity

  Nancy Cooke

  Stephen Liebhaber

  Yugong Ho

  Molecular and Cellular Biology

  For many mammalian genes

  initiation of transcription during embryonic development must be subsequently sustained over extensive periods of adult life. It remains unclear whether maintenance of gene expression reflects the same set of pathways as are involved in initial gene activation. The human pituitary growth hormone (hGH-N) locus is activated in the differentiating somatotrope midway through embryogenesis by a multicomponent locus control region (LCR). DNase I-hypersensitive site I (HSI) of the LCR is essential to full developmental activation of the hGH-N locus. Here we demonstrate that conditional deletion of HSI from the active hGH locus in the adult pituitary effectively silences hGH-N expression. Analyses of chromatin structure and locus positioning demonstrate that a specific subset of the HSI functions active in the embryo retain their HSI dependence in the adult pituitary. These functions sustain engagement of the hGH locus with polymerase II (Pol II) factories

  histone acetylation at the hGH-N promoter

  and looping of the LCR to its target promoter. These data reveal that HSI is essential to both the maintenance and the initiation phases of gene expression. These observations contribute to our mechanistic understanding of how stable patterns of mammalian gene expression are established in a terminally differentiated cell.

  Distinct chromatin configurations regulate the initiation and the maintenance of hGH gene expression

  Obesity is a significant public health concern and a central component of metabolic syndrome. An important scientific and clinical aspect of obesity is the considerable evidence for its developmental origins in the periconceptual and perinatal environment

  indicating that the susceptibility to obesity can be mediated by environmental inputs that stably alter metabolic outcomes. These observations reflect the function of an active epigenetic mechanism that can biochemically program gene expression through DNA and histone modification

  a hypothesis supported by studies in animal models and human cohorts showing strong correlations between early developmental exposures

  chromatin modifications

  and obesity. Consistent with the central role of the hypothalamus in regulating energy balance

  many of the epigenetic effects of obesogenic exposures are targeted to the orexigenic and anorexigenic functions of the hypothalamic feeding center

  and their ability to properly regulate feeding behavior

  energy expenditure

  and metabolic flux in peripheral tissues.

  Epigenetics and Obesity

  Retinoids

  natural and synthetic derivatives of vitamin A

  induce cellular changes by activating nuclear retinoic acid receptors (RAR) and retinoid X receptors (RXR). Although the ability of retinoids to govern gene expression is exploited clinically for cancer therapeutics

  the full benefit of retinoid-based strategies is unrealized due to detrimental side effects. Delineating the receptors that prompt cellular outcomes is critical to advancing retinoid-based approaches. Here

  we identify the receptors that evoke multiple responses in cutaneous T-cell lymphoma (CTCL). The data demonstrate that RARalpha drives integrin beta7-dependent adhesion and CCR9-mediated chemotaxis in CTCL cells. Of note

  concomitant activation of RARalpha and RXR nuclear receptors yielded synergistic increases in adhesion and migration at concentrations where single agents were ineffective. As the established paradigm of retinoid action in CTCL is apoptosis and growth arrest

  the role of RARalpha/RXR in these events was studied. As with adhesion and migration

  RARalpha/RXR synergism prompted apoptosis and dampened CTCL cell proliferation. Strikingly

  RARalpha/RXR synergism induced responses from CTCL cell lines previously reported to be unresponsive to retinoids. These data provide a novel framework that may further refine a proven CTCL therapy.

  RARalpha/RXR synergism potentiates retinoid responsiveness in cutaneous T-cell lymphoma cell lines

  Nancy Cooke

  Stephen Liebhaber

  The human GH (hGH) gene cluster is regulated by a remote 5' locus control region (LCR). HSI

  an LCR component located 14.5 kb 5' to the hGH-N promoter

  constitutes the primary determinant of high-level hGH-N activation in pituitary somatotropes. HSI encompasses an array of three binding sites for the pituitary-specific POU homeodomain factor Pit-1. In the present report we demonstrate that all three Pit-1 sites in the HSI array contribute to LCR activity in vivo. Furthermore

  these three sites as a unit are fully sufficient for position-independent and somatotrope-restricted hGH-N transgene activation. In contrast

  the hGH-N transgene is not activated by Pit-1 sites native to either the hGH-N or rat (r)GH gene promoters. These findings suggest that the structures of the Pit-1 binding sites at HSI specify distinct chromatin-dependent activities essential for LCR-mediated activation of hGH in the developing pituitary.

  Specification of unique Pit-1 activity in the hGH locus control region

  Ross Hardison

  Randall Miller

  Timothy Zeigler

  Susan Yost

  Magdelena James-Pederson

  Journal of Biological Chemistry

  Despite their descent from a common ancestral gene and the requirement for coordinated

  tissue-specific regulation

  the alpha- and beta-globin genes in many mammals are regulated in distinctly different ways. Unlike the beta-globin gene

  the rabbit alpha-globin gene is transiently expressed at a high level without an added enhancer in transfected erythroid and non-erythroid cells. By examining a series of alpha/beta fusion genes

  we show that internal sequences of the rabbit alpha-globin gene (within the first two exons and introns) are required along with the 5' flank for this enhancer-independent expression. Furthermore

  deletion of the introns of the alpha-globin gene

  or replacement by introns of the beta-globin gene

  results in severely decreased expression of the transfecting genes. Hybrid constructs between segments of the alpha-globin gene and a luciferase gene confirm that internal alpha-globin sequences are needed for high level production of RNA in transfected cells. The flanking and internal sequences implicated in regulation of the rabbit alpha-globin gene coincide with a prominent CpG-rich island and may comprise an extended promoter (including both flanking and intragenic sequences) that is active in transfected cells without an enhancer.

  Flanking and intragenic sequences regulating the expression of the rabbit alpha-globin gene

  Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies

  the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). These findings implicate BAY60 as a potential pharmacotherapeutic agent against aberrant ASM growth disorders such as CAD and also establish a unique mechanism through which VASP controls PKG activity.

  Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation

  Stephen Liebhber

  Nancy Cooke

  Sylvia Asa

  The human growth hormone gene cluster is composed of five closely related genes. The 5'-most gene in the cluster

  hGH-N

  is expressed exclusively in somatotropes and lactosomatotropes of the anterior pituitary. Although the hGH-N promoter contains functional binding sites for multiple transcription factors

  including Sp1

  Zn-15

  and Pit-1

  predictable and developmentally appropriate expression of hGH-N transgenes in the mouse pituitary requires the presence of a previously characterized locus control region (LCR) composed of multiple chromatin DNase I hypersensitive sites (HS). LCR determinant(s) necessary for hGH-N transgene activation are largely conferred by two closely spaced HS (HS I

  II) located 14.5 kilobase pairs upstream of the hGH-N gene. The region sufficient to mediate this activity has recently been sublocalized to a 404-base pair segment of HS I

  II (F14 segment). In the present study

  we identified multiple binding sites for the pituitary POU domain transcription factor Pit-1 within this segment. Using a transgenic founder assay

  these sites were shown to be required for high level

  position-independent

  and somatotrope-specific expression of a linked hGH-N transgene. Because the Pit-1 sites in the hGH-N gene promoter are insufficient for such gene activation in vivo

  these data suggested a unique chromatin-mediated developmental role for Pit-1 in the hGH LCR.

  Pit-1 binding sites in the somatotrope-specific HS I

  II region of the hGH LCR are essential for in vivo hGH-N gene activation

  SCOPE: Dietary fat composition can modulate gene expression in peripheral tissues in obesity. Observations of the dysregulation of growth hormone (GH) in obesity indicate that these effects extend to the hypothalamic-pituitary (H-P) axis. The authors thus determine whether specific high fat (HF) diets influence the levels of Gh and other key gene transcripts in the H-P axis. METHODS AND RESULTS: C57BL/6 mice are fed a lean control diet or a HF diet in the absence or presence of OA

  EPA

  or DHA ethyl esters. Comparative studies are conducted with menhaden fish oil. The HF diet lowered pituitary Gh mRNA and protein levels

  and cell culture studies reveal that elevated insulin and glucose can reduce Gh transcripts. Supplementation of the HF diet with OA

  EPA

  DHA

  or menhaden fish oil do not improve pituitary Gh levels. The HF diet also impaired the levels of additional genes in the pituitary and hypothalamus

  which are selectively rescued with EPA or DHA ethyl esters. The effects of EPA and DHA are more robust relative to fish oil. CONCLUSION: A HF diet can affect H-P axis transcription

  which can be mitigated in some genes by EPA and DHA

  but not fish oil in most cases.

  High Fat Diet Dysregulates Hypothalamic-Pituitary Axis Gene Expression Levels which are Differentially Rescued by EPA and DHA Ethyl Esters

  Vesna Karschner

  Holly Jefferson

  Katherine Hogan

  Journal of Molecular Biology

  The POU domain transcription factor Pit-1 is expressed in somatotropes

  lactotropes

  and thyrotropes of the anterior pituitary. Pit-1 is essential for the establishment of these lineages during development and regulates the expression of genes encoding the peptide hormones secreted by each cell type

  including the growth hormone gene expressed in somatotropes. In contrast to rodent growth hormone loci

  the human growth hormone (hGH) locus is regulated by a distal locus control region (LCR)

  which is required in cis for the proper expression of the hGH gene cluster in transgenic mice. The hGH LCR mediates a domain of histone acetylation targeted to the hGH locus that is associated with distal hGH-N activation

  and the discrete determinants of this activity coincide with DNaseI hypersensitive site (HS) I of the LCR. The identification of three in vitro Pit-1 binding sites within the HS-I region suggested a model in which Pit-1 binding at HS-I initiates the chromatin modification mechanism associated with hGH LCR activity. To test this hypothesis directly and to determine whether Pit-1 expression is sufficient to confer hGH locus histone acetylation and activate hGH-N transcription from an inactive locus

  we expressed Pit-1 in nonpituitary cell types. We show that Pit-1 expression established a domain of histone hyperacetylation at the LCR and hGH-N promoter in these cells similar to that observed in pituitary chromatin. This was accompanied by the activation of hGH-N transcription and an increase in intergenic and CD79b transcripts proximal to HS-I. These effects were coincident with Pit-1 occupancy at HS-I and the hGH-N promoter and were observed irrespective of the basal histone modification status of HS-I in the heterologous cell line. These findings are consistent with a role for Pit-1 as an initiating factor in hGH locus activation during somatotrope ontogeny

  acting through binding sites at HS-I of the hGH LCR.

  Expression of Pit-1 in non-somatotrope cell lines induces human growth hormone locus control region histone modification and hGH-N transcription

  Fred Bertrand

  Karlene Hewan-Lowe

  Anne Kellog

  Jarrett Whelan

  Journal of Cellular Biochemistry

  Prostate tumorigenesis is associated with loss of PTEN gene expression. We and others have recently reported that PTEN is regulated by Notch-1 signaling. Herein

  we tested the hypothesis that alterations of the Notch-1 signaling pathway are present in human prostate adenocarcinoma and that Notch-1 signaling regulates PTEN gene expression in prostate cells. Prostate adenocarcinoma cases were examined by immunohistochemistry for ligand cleaved (activated) Notch-1 protein. Tumor foci exhibited little cleaved Notch-1 protein

  but expression was observed in benign tissue. Both tumor and benign tissue expressed total (uncleaved) Notch-1. Reduced Hey-1 expression was seen in tumor foci but not in benign tissue

  confirming loss of Notch-1 signaling in prostate adenocarcinoma. Retroviral expression of constitutively active Notch-1 in human prostate tumor cell lines resulted in increased PTEN gene expression. Incubation of prostate cell lines with the Notch-1 ligand

  Delta

  resulted in increased PTEN expression indicating that endogenous Notch-1 regulates PTEN gene expression. Chromatin immunoprecipitation demonstrated that CBF-1 was bound to the PTEN promoter. These data collectively indicate that defects in Notch-1 signaling may play a role in human prostate tumor formation in part via a mechanism that involves regulation of the PTEN tumor suppressor gene.

  Notch-1 signaling is lost in prostate adenocarcinoma and promotes PTEN gene expression

  Xiaofei Chen

  Holly Jefferson

  Alona Nakonechnaya

  Journal of Cellular Biochemistry

  The prostate gland is regulated by multiple hormones and growth factors that may also affect prostate tumorigenesis. Growth hormone (GH) contributes to prostate development and function

  but the direct effects of GH on prostate cancer cells are not well understood. The expression of endogenous GH in prostate cancer cell lines has also been observed

  suggesting the potential for an effect of autocrine GH. In the present study

  we measure the levels of GH and GH receptor (GHR) mRNA in multiple prostate cancer and normal prostate-derived cell lines

  and compare the effects of exogenous and autocrine GH on LNCaP prostate cancer cell proliferation and apoptosis

  and the associated signal transduction pathways. We found that GHR and GH expression were higher in the prostate cancer cell lines

  and that exogenous GH increased LNCaP cell proliferation

  but had no effect on apoptosis. In contrast

  autocrine GH overexpression reduced LNCaP cell proliferation and increased apoptosis. The distinct actions of exogenous and autocrine GH were accompanied by differences in the involvement of GHR-associated signal transduction pathways

  and were paralleled by an alteration in the subcellular localization of GHR

  in which autocrine GH appeared to sequester GHR in the Golgi and endoplasmic reticulum. This alteration of GHR trafficking may underlie a distinct mode of GH-mediated signaling associated with the effect of autocrine GH. These findings clarify the potential effects of GH on prostate cancer cell function

  and indicate that the activity of autocrine GH may be distinct from that of endocrine GH in prostate cancer cells.

  Differential effects of exogenous and autocrine growth hormone on LNCaP prostate cancer cell proliferation and survival

  Nancy Cooke

  Stephen Liebhaber

  Yugong Ho

  Activation of the human growth hormone (hGH-N) gene in pituitary somatotropes is mediated by a locus control region (LCR). This LCR is composed of DNase I-hypersensitive sites (HS) located -14.5 kb to -32 kb relative to the hGH-N promoter. HSI

  at -14.5 kb

  is the dominant determinant of hGH-N expression and is essential for establishment of a 32-kb domain of histone acetylation that encompasses the active hGH locus. This activity is conferred by three binding sites for the POU domain transcription factor Pit-1. These Pit-1 elements are sufficient to activate hGH-N expression in the mouse pituitary. In contrast

  Pit-1 sites at the hGH-N promoter are consistently unable to mediate similar activity. In the present study

  we demonstrate that the functional difference between the promoter-proximal and the HSI Pit-1 binding sites can be attributed in part to a single base difference. This base affects the conformation of the Pit-1/DNA complex

  and reciprocal exchange of the divergent bases between the two sets of Pit-1 elements results in a partial reversal of their transgenic activities. These data support a model in which the Pit-1 binding sites in the hGH LCR allosterically program the bound Pit-1 complex for chromatin activating functions.

  A single base difference between the Pit-1 binding sites at the hGH promoter and locus control region specifies distinct Pit-1 conformations and functions

  Alona O. Nakonechnaya

  Endocrine Research

  Purpose: Prostate cancer cells are responsive to multiple hormones and growth factors that can affect cell function. These effects may include modulating cell proliferation and apoptosis

  but the ability to impinge on the metastatic potential of prostate cancer cells by affecting cell motility should also be considered

  as prostate tumor metastasis correlates with limited therapeutic options and poor prognosis. Human growth hormone (hGH) can affect the growth and survival of prostate cancer cells

  but the effect of hGH on prostate cancer cell motility is unknown. In the present study

  the potential for exogenous and autocrine hGH to directly affect prostate cancer cell motility was addressed. Materials and Methods: The effects of exogenous and autocrine hGH on the chemokinesis and chemotaxis of LNCaP prostate cancer cells were tested using cell monolayer wound healing and Boyden chamber invasion assays. The signaling pathways underlying these effects were resolved with chemical inhibitors and the correlation with cytoskeletal actin reorganization evaluated microscopically by staining cells with fluor-conjugated phalloidin. Results: Both exogenous and autocrine hGH augmented the migration and invasion of LNCaP cells

  and hGH itself acted as a chemoattractant. This activity was dependent upon the STAT5

  MEK1/2 and PI3K signaling pathways

  and was accompanied by an alteration in cellular actin organization. Conclusions: hGH may enhance the metastatic potential of prostate cancer cells

  both as a stimulant of cellular motility and invasiveness and as a chemoattractant.

  Growth hormone enhances LNCaP prostate cancer cell motility

  Joseph Houmard

  Todd Weber

  Monica Hubal

  Naomi Rowland

  Kai Zou

  Carol Witczak

  Jeffrey Brault

  Jill Maples

  Physiological Genomics

  The skeletal muscle of obese individuals exhibits an impaired ability to increase the expression of genes linked with fatty acid oxidation (FAO) upon lipid exposure. The present study determined if this response could be attributed to differential DNA methylation signatures. RNA and DNA were isolated from primary human skeletal muscle cells (HSkMC) from lean and severely obese women following lipid incubation. mRNA expression and DNA methylation were quantified for genes that globally regulate FAO [PPARγ coactivator (PGC-1α)

  peroxisome proliferator-activated receptors (PPARs)

  nuclear respiratory factors (NRFs)]. With lipid oversupply

  increases in NRF-1

  NRF-2

  PPARα

  and PPARδ expression were dampened in skeletal muscle from severely obese compared with lean women. The expression of genes downstream of the PPARs and NRFs also exhibited a pattern of not increasing as robustly upon lipid exposure with obesity. Increases in CpG methylation near the transcription start site with lipid oversupply were positively related to PPARδ expression; increases in methylation with lipid were depressed in HSkMC from severely obese women. With severe obesity

  there is an impaired ability to upregulate global transcriptional regulators of FAO in response to lipid exposure. Transient changes in DNA methylation patterns and differences in the methylation signature with severe obesity may play a role in the transcriptional regulation of PPARδ in response to lipid. The persistence of differential responses to lipid in HSkMC derived from lean and obese subjects supports the possibility of stable epigenetic programming of skeletal muscle cells by the respective environments.

  Lipid exposure elicits differential responses in gene expression and DNA methylation in primary human skeletal muscle cells from severely obese women

  Joseph Houmard

  Todd Weber

  Monica Hubal

  Sanghee Park

  Carol Witczak

  Jeffrey Brault

  Jill Maples

  American Journal of Physiology - Endocrinology & Metabolism

  The ability to increase fatty acid oxidation (FAO) in response to dietary lipid is impaired in the skeletal muscle of obese individuals

  which is associated with a failure to coordinately upregulate genes involved with FAO. While the molecular mechanisms contributing to this metabolic inflexibility are not evident

  a possible candidate is carnitine palmitoyltransferase 1B (CPT1B)

  which is a rate-limiting step in FAO. The present study was undertaken to determine if the differential response of skeletal muscle CPT1B gene transcription to lipid between lean and severely obese subjects is linked to epigenetic modifications (DNA methylation and histone acetylation) that impact transcriptional activation. In primary human skeletal muscle cultures the expression of CPT1B was blunted in severely obese women compared to their lean counterparts in response to lipid

  which was accompanied by changes in CpG methylation

  H3/H4 histone acetylation

  and peroxisome proliferator-activated receptor δ (PPARδ) and hepatocyte nuclear factor 4α (HNF4α) transcription factor occupancy at the CPT1B promoter. Methylation of specific CpG sites in the CPT1B promoter that correlated with CPT1B transcript level blocked the binding of the transcription factor USF

  suggesting a potential causal mechanism. These findings indicate that epigenetic modifications may play important roles in the regulation of CPT1B in response to a physiologically relevant lipid mixture in human skeletal muscle

  a major site of fatty acid catabolism

  and that differential DNA methylation may underlie the depressed expression of CPT1B in response to lipid

  contributing to the metabolic inflexibility associated with severe obesity.

  Differential Epigenetic and Transcriptional Response of the Skeletal Muscle Carnitine Palmitoyltransferase 1B (CPT1B) Gene to Lipid Exposure with Obesity.

  Ross Hardison

  In contrast to other globin genes

  the human and rabbit alpha-globin genes are expressed in transfected erythroid and nonerythroid cells in the absence of an enhancer. This enhancer-independent expression of the alpha-globin gene requires extensive sequences not only from the 5' flanking sequence but also from the intragenic region. However

  the features of these internal sequences that are responsible for their positive effect are unclear. We tested several possible determinants of this activity. One possibility is that a previously identified array of discrete binding sites for known and potential regulatory proteins within the alpha-globin gene comprise an intragenic enhancer specific for the alpha-globin promoter

  but directed rearrangements of the sequences show that this is not the case. Alternatively

  the promoter may extend into the gene

  with the function of the discrete binding sites being dependent on maintenance of their proper positions and orientations relative to the 5' flanking sequence. However

  the positive effects observed in gene fusions do not localize to a discrete region of the alpha-globin gene and the results of internal deletions and point mutations argue against a required role of the targeted discrete binding sites. A third possibility is that the CpG island

  which includes both the 5' flanking and intragenic regions associated with the positive activity

  may itself have a more general effect on expression in transfected cells. Indeed

  we show that the size of the CpG island in constructs correlates with the level of gene expression. Furthermore

  the alpha-globin promoter is more active in the context of a previously inactive CpG island than in an A+T-rich context

  showing that the CpG island provides an environment more permissive for expression. These effects are seen only after integration

  suggesting a possible mechanism at the level of chromatin structure.

  CpG islands from the alpha-globin gene cluster increase gene expression in an integration-dependent manner

  The hypothalamic-pituitary (H-P) axis integrates complex physiological and environmental signals and responds to these cues by modulating the synthesis and secretion of multiple pituitary hormones to regulate peripheral tissues. Prostaglandins are a component of this regulatory system

  affecting multiple hormone synthesis and secretion pathways in the H-P axis. The implications of these actions are that physiological processes or disease states that alter prostaglandin levels in the hypothalamus or pituitary can impinge on H-P axis function. Considering the role of prostaglandins in mediating inflammation

  the potential for neuroinflammation to affect H-P axis function in this manner may be significant. In addition

  the mitigating effects of n-3 polyunsaturated fatty acids (n-3 PUFA) on the inflammation-associated synthesis of prostaglandins and their role as substrates for pro-resolving lipid mediators may also include effects in the H-P axis. One context in which neuroinflammation may play a role is in the etiology of diet-induced obesity

  which also correlates with altered pituitary hormone levels. This review will survey evidence for the actions of prostaglandins and other lipid mediators in the H-P axis

  and will address the potential for obesity-associated inflammation and n-3 PUFA to impinge on these mechanisms.

  Prostaglandins and n-3 polyunsaturated fatty acids in the regulation of the hypothalamic-pituitary axis

  Stephen Liebhaber

  Nancy Cooke

  Expression of the human growth hormone (hGH-N) transgene in the mouse pituitary is dependent on a multicomponent locus control region (LCR). The primary determinant of hGH LCR function maps to the pituitary-specific DNase I hypersensitive sites (HS) HSI

  II

  located 15 kb 5' to the hGH-N gene. The mechanism by which HSI

  II mediates long-distance activation of the hGH locus remains undefined. Matrix attachment regions (MARs) comprise a set of AT-rich DNA elements postulated to interact with the nuclear scaffold and to mediate long-distance interactions between LCR elements and their target promoters. Consistent with this model

  sequence analysis strongly predicted a MAR determinant in close proximity to HSI

  II. Surprisingly

  cell-based analysis of nuclear scaffolds failed to confirm a MAR at this site

  and extensive mapping demonstrated that the entire 87 kb region encompassing the hGH LCR and contiguous hGH gene cluster was devoid of MAR activity. Homology searches revealed that the predicted MAR reflected the recent insertion of a LINE 3'-UTR segment adjacent to HSI

  II. These data point out discordance between sequence-based MAR predictions and in vivo MAR function and predict a novel MAR-independent mechanism for long-distance activation of hGH-N gene expression.

  The human growth hormone locus control region mediates long-distance transcriptional activation independent of nuclear matrix attachment regions

  East Carolina University Division of Health Sciences

  Howard Hughes Medical Institute

  University of Pennsylvania

  Greenville

  NC

  Graduate and Medical education; Biomedical research; Graduate and undergraduate research mentorship

  Assistant Professor of Biochemistry & Molecular Biology

  East Carolina University Division of Health Sciences

  Graduate and Medical education; Biomedical research; Graduate and undergraduate research mentorship

  East Carolina University Division of Health Sciences

  University of Pennsylvania

  Philadelphia

  PA

  Biomedical research

  Postdoctoral Fellow

  Philadelphia

  PA

  Biomedical research

  Research Associate

  Howard Hughes Medical Institute

  Philadelphia

  PA

  Biomedical research; Graduate and undergraduate research mentorship

  Instructor of Genetics

  University of Pennsylvania

  The Endocrine Society

  American Society for Biochemistry and Molecular Biology

  Association of Biochemistry Course Directors