Molecular Mechanisms of Regulation of SLC11A1 Gene Expression By: Yong Zhong Xu Department of Medicine, Division of Experimental Medicine McGill University, Montreal May 2013 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy Copyright 2013, Yong Zhong Xu 1 TABLE OF CONTENTS Abstract………………………………………………………………………………………………………...5 Résume………………………………………………………………………………….........................8 Acknowledgements………………………………………………………………………………………11 Preface & Contribution of Authors……………………………………………………………….13 Abbreviations………………………………………………………………………………………………15 List of Figures and Tables……………………………………………………………………………..18 Rationale, Objectives and General Outline of the Research………………………...21 Chapter 1: Literature review……………………………………………………………………….29 1.1 Genetics of infectious disease…………………………………………………………….31 1.2 SLC11A1 ……………………………………………………………………………………….…..34 1.2.1 Identification…………………………………………………………………………..…34 1.2.2 SLC11A1 Structure…………………………………………………………………..…37 1.2.2.1 Gene structure…………………………………………………………..…….37 1.2.2.2 Protein Structure…………………………………………………………..…38 1.2.3 Expression and Localization……………………………………………………..…42 1.2.4 SLC11A1 Function …………………………………………………………………..…..43 1.2.4.1 Regulation of macrophage activation ………………………………….44 1.2.4.2 Divalent cation transport …………………………………………………....45 1.2.4.3 SLC11A1 and Resistance to infection …………………………………..51 1.2.4.4 SLC11A1 polymorphism and disease susceptibility ………........55 1.2.5 Orthologs ……………………………………………………………………………….……58 2 1.3 Regulation of SLC11A1 gene expression ………………………………………...59 1.3.1 Overview ………………………………………………………………………………….59 1.3.2 Induction of Slc11a1 (nramp1) expression …………………………….…59 1.3.3 Regulation of Slc11a1/SLC11A1 gene expression at transcriptional Level ………………………………………………………………………………………..60 1.3.4 Regulation of Slc11a1/SLC11A1 gene expression by mRNA stability ……………………………………………………………………………………………….…62 1.3.5 Regulation of Slc11a1 subcellular localization ……………………….…64 Chapter 2: Nuclear Translocation of ‐actin is Involved in Transcriptional Regulation of SLC11A1 Gene during Macrophage Differentiation of HL‐60 Cells ………………………………………………………………....................………..66 Abstract …………………………………………………………………………………………………...67 Introduction ……………………………………………………………………………………………..68 Materials and Methods …………………………………………………………………………….71 Results………………………………………………………………………………………………………78 Discussion ……………………………………………………………………………………………...104 Acknowledgements ……………………………………………………………………………..…114 Supplementary Materials ……………………………………………………………………....115 Chapter 3: Recruitment of SWI/SNF complex is required for transcriptional activation of SLC11A1 gene during macrophage differentiation of HL‐60 cells ………………………………………………………………………………………….124 Abstract ………………………………………………………………………………………………..125 3 Introduction ………………………………………………………………………………………….….126 Materials and Methods …………………………………………………………………………….130 Results ……………………………………………………………………………………………………..139 Discussion …………………………………………………………………………………………………167 Acknowledgements ………………………………………………………………………………….172 Chapter 4: Src family kinase activity is involved in tyrosine phosphorylation and subcellular localization of SLC11A1 in macrophages …………………………………..173 Abstract …………………………………………………………………………………………………....174 Introduction …………………………………………………………………………………………..…175 Materials and Methods …………………………………………………………………………....178 Results ……………………………………………………………………………………………………….186 Discussion ……………………………………………………………………………………………….…213 Acknowledgements …………………………………………………………………………………. 220 Chapter 5: Summary and General Discussion …………………………………………….221 5.1 Summary ………………………………………………………………………………………….222 5.2 Discussion ………………………………………………………………………………………..224 5.3 Conclusion ………………………………………………………………………………………..231 Chapter 6: Claims to Originality ………………………………………………………………….233 Reference List …………………………………………………………………………………………….235 Appendix I: Table S2.3 β‐actin target genes identified by ChIP‐on‐chip in HL‐60 cells left untreated or treated with PMA for 48 hrs …………………………………….265 Appendix II: List of other published papers and book chapters ………………..293 Appendix III: Research Compliance Certificates …………………………………………295 4 ABSTRACT Solute carrier family 11 member 1 protein (SLC11A1), also known as natural resistance‐associated macrophage protein 1 (NRAMP1), plays an important role in the host immune defense and inflammatory response. It is a highly conserved transmembrane protein which transports divalent metal cations in a proton‐ dependent manner. It regulates iron homeostasis in macrophages and exerts pleiotropic effects on macrophage activation. In mice, natural resistance or susceptibility to a range of intracellular pathogens is controlled by the Slc11a1 gene. In human, genetic polymorphisms of the SLC11A1 gene have been shown to be associated with a susceptibility to a variety of infectious and autoimmune diseases. The expression of the SLC11A1 gene is strictly regulated during myeloid differentiation. The human promyelocytic leukemia cell lines such as HL‐60 and U937 cells are useful models to study the regulation of SLC11A1 gene expression during experimentally induced granulocytic, monocytic, or macrophage‐like differentiation. Herein, we demonstrated that during the PMA‐induced differentiation of HL‐60 cells and human monocytes toward macrophages, ‐ actin translocates from the cytoplasm to the nucleus where it is associated with RNA polymerase II and binds to the promoter of the SLC11A1 gene. ‐actin knockdown inhibits the SLC11A1 promoter‐driven transcription, and neutralization of nuclear actin by in vivo microinjection of antibodies against ‐ 5 actin into nuclei significantly blocks the expression of SLC11A1 mRNA. Further studies revealed that an AP‐1‐like element present in the proximal region of the SLC11A1 gene promoter is essential for PMA‐induced transcriptional activation of this gene. β‐actin, as a subunit of the SWI/SNF complex, and another subunit BRG1 are associated with the transcription factor ATF‐3 and are recruited to the AP‐1 like element in an ATF‐3‐dependant manner . ATF‐3 cooperates with BRG1 and β‐actin to activate the SLC11A1 promoter. Furthermore, a proximal (GT/AC)n repeat (t(gt)5ac(gt)5ac(gt)9g) region adjacent to the AP‐1‐like element is converted into a Z‐DNA structure in response to PMA treatment, and BRG1 is involved in this process. Our results suggest that recruitment of the SWI/SNF complex initiates Z‐DNA formation and subsequently helps to transactivate the SLC11A1 gene. Previous studies have shown that SLC11A1 is extensively glycosylated and phosphorylated, and is localized at the membrane of late endosomes/lysosomes in macrophages. The present study revealed that SLC11A1 is tyrosine‐phosphorylated during the differentiation of U937 cells into macrophages induced by PMA. Using the kinase inhibitor PP2 and RNA interference experiments, we demonstrated that Src family kinases including c‐ Src are required for the tyrosine phosphorylation of SLC11A1 protein. In vitro phosphorylation assays showed that SLC11A1 is a direct substrate for active c‐Src kinase. Furthermore, tyrosine 15 is identified as the tyrosine phosphorylation site by Src family kinases and phosphorylation of tyrosine 15 modulates SLC11A1‐mediated nitric oxide production. We also showed that the Src family 6 kinases including c‐Src are also involved in lysosomal targeting of SLC11A1. These results suggest an important role of Src family kinases in subcellular localization and function of SLC11A1 in macrophages. Overall, our studies contributed important information on the regulation of expression of SLC11A1 in macrophages and its role in regulation of macrophage functions. 7 Résumé La protéine « Solute carrier family 11 member 1 » (SLC11A1), aussi connue sous le nom « natural resistance associated macrophage protein 1 » (NRAMP1), jour un rôle important dans la défense immunitaire et réponse inflammatoire de l'hôte. C’est une protéine transmembranaire hautement conservée qui transporte des cations divalents métalliques d’une manière dépendent des protons. Elle régularise l’homéostasie du fer dans les macrophages et a des effets pléiotropiques sur l’activation de ces cellules. Chez les souris, le gène Slc11a1 contrôle la resistance naturelle ou la susceptibilité aux pathogènes intracellulaires. Chez les humains, les polymorphismes génétiques de SLC11A1 sont associés à une susceptibilité à une variété de maladies infectieuses ou auto‐ immunitaires. L’expression du gène SLC11A1 est strictement régularisée pendant la différenciation myéloïde. Les lignées cellulaires humaines dérivées de la leucémie aiguë promyélocytaire, telles que la HL‐60 et l’U937, sont des modèles utiles pour étudier le contrôle de l’expression du gène SLC11A1 pendant la différenciation de type granulocytaire, monocytaire ou de macrophage induite expérimentalement. Ici, nous avons démontré que durant la différenciation induite par PMA des cellules HL‐60 et des monocytes humains aux macrophages, β‐actine passe du cytoplasme au noyau où il s’associe avec l’ARN polymérase II et se fixe sure le promoteur du gène SLC11A1. Le « knock‐down » de la β‐actine inhibe la transcription menée par le promoteur de gène SLC11A1. Dans le noyau, 8 l’expression de l’ARN de SLC11A1 est bloqué significativement en neutralisant l’actine par la microinjection in vivo des anticorps contre β‐actine. Autres études ont démontré qu’un élément « semblable à AP‐1 » est présent dans la région proximale du promoteur de SLC11A1 et celui‐ci est essentiel