A systems biology approach to elucidating the frequency decoding mechanism governing differential mammalian gonadotropin subunit gene expression

A SYSTEM BIOLOGY APPROACH TO ELUCIDATING THE GnRH FREQUENCY DECODING MECHANISM THAT GOVERNS DIFFERENTIAL EXPRESSION OF THE GONADOTROPIN-SUBUNIT GENES STEFAN LIM B.Sc(Hons.), Edin. U A THESIS SUBMITTED IN ACCORDANCE WITH THE REQUIREMENTS OF THE NATIONAL UNIVERSITY OF SINGAPORE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Acknowledgments It would be hard to envision myself completing this arduous journey of half-a-decade without the tremendous help and encouragement of the following people: My deceased father, who before he passed on secretly told my mother that I would secure this scholarship to commence graduate studies, who believed I could embark on this treacherous journey and make it. My mother, who upheld and continued the convictions of my father, and encouraged me throughout this period; if nothing else, silently praying for strength and perseverance for me. My wife, who has remained patient and understanding throughout this time, enduring lengthy periods of loneliness when through the force of circumstances, I have had to devote more time to research than to her. Dr. Guna, who made it possible for me to this PhD, by first accepting me into the M.Sc in Bioinformatics programme, and then recommending me to A-Star for the award of the Ph.D scholarship. If the former hadn’t happen, I would never have entered the beautiful world of Biology i Dr. Philippa, whom I will always maintain as the best person who could ever have supervised me, who took every risk imaginable in accepting me into her lab as an ignorant intern at first, and then later, as her student. Moreover, for the last half-a-year of my candidature, when my stipend had dried up, she gave me employment in the lab, so that I would never have to go hungry even for a day. I will never cease to respect and marvel at her trust in my non-abilities, which she constantly sees as opportunities for personal growth and fulfillment, and to be grateful to her for the one memorable visit to Israel, the most beautiful country on earth. She is truly God-sent. Prof. Zvi Naor, who has inspired me a great deal not only through his published work in this field of gonadotropin gene regulation, but also through active discussions with him during his visits to Singapore, as well as during my visit to Israel. He embodies all of what great scientists ought to have - intelligence, drive, fantasy and an aura of humanity, humility and congeniality. Mingshi, who mentored me and taught me so patiently every aspect of experimental Biology, who taught me the beauty of life, and who is the sole reason why I have chosen to pursue a Ph.D in this field and in this lab. Stella, who was my dearest friend and god sister, and had been the constant inspiration in my life, however hard and trying times might have been. She taught me the simple truths of selfless love and friendship, and that it was not shameful nor cowardly to cry when things surrounding me became overwhelmingly difficult to bear. In more ways than one, and as only she would comprehend, I owe my continued existence to her. ii Kathy, who became my friend very late on in my PhD career, and when she was about to leave Singapore for France to pursue her own academic dreams. She epitomizes everything of a great scientist-to-be, and is probably one of the very few people in my life who wouldn’t mind talking science with me on the subway, all the way home. She re-kindled my interest in the French language - good or bad - it is not a worthless skill, at the very least. Andrea and Serena, who have been inseparable in their friendship and inseparable in working their good deeds and charm. Thank you for the little card you gave me before you left our lab, bearing a message that reminded me for the remainder of my time in this lab that clearing trash and dirty bottles every so often was not a thankless task after all. Sue Yuan, who was someone I tried to encourage all through her period of sorrow, but ended up being encouraged by her fortitude and experiences. Thank you for being such a dear friend, and for the mince pies you brought back from England. Members of Philippa’s Lab, some of whom have out-stayed me, while others haven’t. Regardless, each one of them has contributed no small part to my reaching the end, and has made the pain of each experimental failure a little less. Liu Ping, who helped me much with all the experiments involving FCCS and live cell imaging. Keng Hwee, who has at times played the role of devil’s advocate, and at other times, the author’s advocate. Whichever role he assumed, he did it better than anyone else. A*star, who funded this research project and also my studies. iii NGS, who supported me administratively throughout the course of my studies. Celine, who came into my life rather unexpectedly, but most timely. Her extraordinary blend of teenage innocence and youthful exuberance worked wonders for an aching heart, tormented by the mistrust of others and the despair of a rejected thesis. She acted as an angel commissioned by God, who appeared, and then disappeared - but who in the few weeks that we shared life together, became my wonderfully adorable child, my sweet and doting kid sister, my most precious friend, and everything else I could and would ever wish for in life. Her charmingly facetious tendencies and insatiable appetite for food and knowledge, were a joy to behold and a pleasure to oblige. She ran alongside me, encouraged me and infused me with just enough strength to complete this final mile. Without her, I most certainly would have given up short of the finishing-line. It is thus only appropriate to reserve my final and most needful word of thanks to an earthly being for her, with whom I was not acquainted when this thesis was first submitted, but fully and endearingly so, by the time it was eventually re-done. God, who is the One I will have to reserve most gratitude and honor for, without whom nothing would have been possible. It was He, who created our amazing universe, and all the science that undergirds the functionality of it all. The pursuit of scientific study is but only a God-given opportunity to try and understand the beauty and wonder of creation. iv Abstract The synthesis of the gonadotropin-subunits is directed by pulsatile gonadotropin-releasing hormone (GnRH) from the hypothalamus, with the frequency of GnRH pulses governing the differential expression of the common α-subunit (αGSU), luteinizing hormone βsubunit (LHβ) and follicle-stimulating hormone β-subunit (FSHβ). In many vertebrate species, levels of these hormones vary quite dramatically throughout their life cycles owing to low levels of GnRH secretion that occur during the juvenile stage, suggesting a native state of gene repression. Preliminary findings point to the actions of histone deacetylases (HDACs) in repressing the gonadotropins. In this study, a system biology approach is taken to unravel the mechanisms for GnRH-frequency decoding and GnRH-induced de-repression of the gonadotropin-subunit genes. Three mitogen-activated protein kinases (MAPKs), ERK1/2, JNK and p38, are known to be contributing uniquely and combinatorially to the expression of each of these subunit genes. Using mathematical modeling and computer simulations, it was found that dual specificity phosphatase (DUSP) regulation of the activity of these MAPKs through negative feedback, forms the basis for decoding the frequency of pulsatile GnRH. Furthermore, a fourth MAPK, ERK5, whose activation kinetics and role in FSHβ gene expression are shown, was found to enhance the preference of FSHβ for low GnRH pulse frequencies. Evidence is presented for ERK5-activation of FSHβ gene expression through Nur77-dependent and independent mechanisms, through interactions with MEF2D. This involves the Ca2+ -activated calcineurin both in activating Nur77 transcription, as well as possibly dephosphorylating Nur77, which is required for its activity. Having established that distinct sets of HDACs repress the two β-subunits, a role for GnRH-activated Ca2+ /calmodulin-dependent protein kinase I (CaMKI) is eluciv dated in the de-repression of the FSHβ gene, which primarily involves phosphorylating certain class IIa HDACs, critical for their nuclear export. Finally, Gem, a negative regulator of calcium L-type channels, is shown to be involved in regulating αGSU expression through influencing ERK1/2 activation in both a Ca2+ -dependent and independent way. These rely on Gem’s ability both to be re-localized to the cytosol upon CaM binding, and to effect cytoskeletal remodeling upon 14-3-3 binding. These findings reveal a complex interplay of signal transducers, transcription factors, and both chromatin- and cytoskeletal-remodeling proteins at different levels to orchestrate the expression of various gonadotropin-subunit genes under the diverse actions of GnRH. vi Contents Acknowledgments i Abstract v Contents xii List of Tables xiii List of Figures xviii Nomenclature xxi Introduction 1.1 The gonadotropic hormones . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 The hypothalamic control of pituitary action . . . . . . . . . . . . 1.1.2 The gonadotropins and their role in reproduction . . . . . . . . . 1.1.3 Gonadotropin-subunit gene regulation at a glance . . . . . . . . . 1.1.4 Understanding gonadotropin-subunit gene expression through the 1.2 use of model cell-lines . . . . . . . . . . . . . . . . . . . . . . . Regulation of gonadotropin expression by pulsatile GnRH . . . . . . . . 1.2.1 subunit gene expression . . . . . . . . . . . . . . . . . . . . . . The GnRH receptor-stimulated network as a frequency decoder . Regulation of gonadotropin expression by calcium . . . . . . . . . . . . 10 1.2.2 1.3 The requirement of pulsatile GnRH for optimal gonadotropin- vii 1.4 1.3.1 The calcium-channel regulator Kir/Gem is induced by GnRH . . 12 1.3.2 Gem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 1.3.3 Both CaM and 14-3-3 localize to lipid rafts in c-raf signaling in the gonadotropes . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Regulation of gonadotropin expression through targeting the chromatin . 17 1.4.1 The fluctuating levels of GnRH at different stages of the vertebrate life cycle reveal a possible natural state of gonadotropinsubunit gene repression . . . . . . . . . . . . . . . . . . . . . . . 1.4.2 Chromatin structure and the repression of the gonadotropin-subunit genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 1.4.3 Histone deacetylases (HDACs) . . . . . . . . . . . . . . . . . . . 19 1.4.4 HDAC activity is involved in the repression of the gonadotropin β-subunit genes, and is overcome by GnRH . . . . . . . . . . . . 1.4.5 1.5 1.6 17 22 Distinct sets of HDACs repress the gonadotropin β-subunit genes in the immature gonadotropes . . . . . . . . . . . . . . . . . . . 23 1.4.6 GnRH activates CaMKI in immature gonadotropes . . . . . . . . 25 1.4.7 Nur77 and MEF2D de-repress the FSHβ gene . . . . . . . . . . . 26 Frequency decoding re-visited: the search for a frequency decoding mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Hypothesis and aims . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 1.6.1 Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 1.6.2 Aims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Experimental Materials and Methods 34 2.1 Cell culture, transfection and treatment . . . . . . . . . . . . . . . . . . . 34 2.1.1 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 2.1.2 Cryo-storage of cells . . . . . . . . . . . . . . . . . . . . . . . . 34 2.1.3 Recovery of cells . . . . . . . . . . . . . . . . . . . . . . . . . . 35 viii 2.1.4 Transfection of cells . . . . . . . . . . . . . . . . . . . . . . . . 35 2.1.5 Chemical treatment of cells . . . . . . . . . . . . . . . . . . . . 35 Plasmid construction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 2.2.1 SiRNA constructs . . . . . . . . . . . . . . . . . . . . . . . . . . 36 2.2.2 Expression vectors . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.2.3 Isolation, verification and plasmid preparation . . . . . . . . . . . 39 RNA extraction and reverse transcriptase PCR . . . . . . . . . . . . . . . 42 2.3.1 RNA isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 2.3.2 First strand cDNA synthesis . . . . . . . . . . . . . . . . . . . . 42 2.3.3 PCR and gel electrophoresis analysis . . . . . . . . . . . . . . . 42 2.4 Luciferase assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 2.5 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 2.6 Whole cell extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 2.7 Co-immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . 45 2.8 Western blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 2.9 Immuno-fluorescence/Confocal microscopy . . . . . . . . . . . . . . . . 47 2.10 Live cell imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 2.11 Fluorescence cross-correlation spectroscopy (FCCS) . . . . . . . . . . . 48 Computational Modeling 50 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 3.1.1 Published models on frequency decoding of GnRH signals . . . . 50 3.1.2 Proposed scheme of model development . . . . . . . . . . . . . . 52 The basic model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 3.2.1 Model development . . . . . . . . . . . . . . . . . . . . . . . . . 55 The intermediate and full models . . . . . . . . . . . . . . . . . . . . . . 58 3.3.1 Model development . . . . . . . . . . . . . . . . . . . . . . . . . 59 Computer simulations and key readouts . . . . . . . . . . . . . . . . . . 65 2.2 2.3 3.2 3.3 3.4 ix [146] Grozinger CM, CA Hassig, and Schreiber SL. 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Dual specificity phosphatase knockout mice show enhanced susceptibility to anaphylaxis but are sensitive to glucocorticoids. Mol. Endocrinol., 21:2663–2671, 2007. 208 At least we don’t have to this ten thousand times!!! Celine Annabelle Yeh 209 [...]... [85] All these calcium-activated PKC isoforms (PKCs), in turn, have important downstream roles in mediating the actions of GnRH on gonadotropin expression [18, 85] 1.2.2.3 Activation of the mitogen-activated protein kinases (MAPKs) One of the notable consequences of calcium release into the cytoplasm and the activation of PKCs is the firing of the three major MAPK cascades, culminating in the activation... known how the king would fall Hey who’s to say you know I might have changed it all And now I’m glad I didn’t know The way it all would end The way it all would go Our lives are better left to chance I could have missed the pain But I’d have had to miss the dance The dance The dance I would have missed the dance The Dance Tom Arata xxii So no one wanted to supervise you immunology? and that’s why you... downstream pathways would also be expected to be similarly activated by the one instance of receptor binding by GnRH Hence, it is crucial to gain a clear understanding of the molecular events that happen upon receptor activation, before a mechanism for GnRH frequency decoding can be purported 1.2.2.1 The GnRH receptor The mammalian GnRH receptor (GnRHR) is composed of 327-328 amino acids and is a member... the α -subunit gene in immature gonadotropes 169 xvii Nomenclature αGSU Glycoprotein α -subunit AP-1 Activator protein 1 BAPTA/AM 1,2-bis-(o-aminophenoxy)ethane-N,N,N’,N’-tetra-acetic acid acetoxymethyl tetraester BMK Big mitogen-activated protein kinase CaM Calmodulin CaMK Ca2+ /calmodulin-dependent protein kinase cAMP 3’-5’-cyclic adenosine monophosphate CoA Coactivator CsA Cyclosporine A DAG Diacylglycerol... activates a number of signaling pathways, including MEK, JNK, ERK1/2, cAMP/PKA, PKC, Ca2+ - and CaM-dependent pathways A number of transcription factors are activated through the phosphorylation by these kinases (Figure adapted from [16].) JNK FSHβ, however, requires all three MAPK pathways The dependence of gonadotropin- subunit gene expression on the MAPKs has two likely implications on the GnRH frequency. .. stimulation of α -subunit and LHβ promoter activity, as well as mRNA levels of all three subunits [110, 111] This suggests that GnRH can act through CaMK to stimulate the gonadotropin- subunit genes CaM has also been implicated in GnRH signaling to ERK1/2 Inhibition of CaM using W-7, a common calmodulin antagonist that binds to calcium-loaded calmodulin (Ca2+ CaM) in place of its normal physiological target... on gonadotropins?!! Celine, aged A* Teen xxiii Chapter 1 Introduction 1.1 1.1.1 The gonadotropic hormones The hypothalamic control of pituitary action The pituitary gland and the hypothalamus are both located within the cranial region (Figure 1.1) The pituitary gland is sometimes known as the ‘master gland’ of the endocrine Figure 1.1: Layout of the human pituitary The hypothalamus is connected to the. .. regulation at a glance Given the importance of the gonadotropins in the vertebrate life cycle, much research in the past two decades has been centered around the regulation of their biosynthesis [8–11] Mouse and rat models, as well as cell-lines, have been employed extensively to look at various aspects of gonadotropin- subunit gene expression For instance, very early on, it was discovered that the pulsatility... of decoding GnRH pulse frequencies, that would give rise to the differential expression of the subunit genes Frequency decoding may be defined as the ability of the gonadotrope cells within the anterior pituitary to recognize different pulse frequencies of GnRH and through its intracellular mechanisms, allow the frequency of GnRH to dictate the predominance in the expression of any subunit gene A number... of the regulators of calcium activity known in other cell types have yet to be studied in the context of the gonadotropes A large-scale microarray screen for genes up-regulated by GnRH in LβT2 cells revealed that the mRNA for Gem (also referred to as Kir), a calcium signaling pathway-associated protein, was increased with GnRH treatment This result was confirmed by quantitative real-time PCR [24] Additionally, . A SYSTEM BIOLOGY APPROACH TO ELUCIDATING THE GnRH FREQUENCY DECODING MECHANISM THAT GOVERNS DIFFERENTIAL EXPRESSION OF THE GONADOTROPIN- SUBUNIT GENES STEFAN LIM B.Sc(Hons.), Edin. U A THESIS. a system biology approach is taken to unravel the mechanisms for GnRH -frequency decoding and GnRH-induced de-repression of the gonadotropin- subunit genes. Three mitogen-activated protein kinases (MAPKs),. recommending me to A- Star for the award of the Ph.D scholarship. If the former hadn’t happen, I would never have entered the beautiful world of Biology i Dr. Philippa, whom I will always maintain as the