THE RING FINGER BINDING PROTEIN IS A NUCLEAR

MEMBRANE PROTEIN THAT INTERACTS

WITH RUSH TRANSCRIPTION FACTORS

by

MALINI MANSHARAMANI, B.S., M.S.

A DISSERTATION

IN

ANATOMY

Submitted to the Graduate Faculty of Texas Tech University Health Sciences Center in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY

Advisory Committee

Beverly S. Chilton (Chairperson) Vaughn H. Lee Curt M. Pfarr Thomas A. Pressley Daniel R. Webster Sandra M. Whelly

Accepted

May, 2001 ACKNOWLEDGEMENTS

There arc a number of people who have contributed immensely to the successful completion of this scientific endeavor. First and foremost, I would like to thank my parents who encouraged me to think big and aim for the stars. I thank them for their unconditional love and understanding and their faith in me and my abilities. With their blessings and prayers, and love and cheer from my sisters, Vinita and Usha, my brother- in-law, Rajesh, my dear nephew, Nitin, my grandmother and all my aunts, uncles, cousins and friends, I embarked on this journey in search of a scientific career. It is here that I acknowledge the tremendous amount of faith, support, patience and encouragement shown to me by my mentor. Dr. Beverly Chilton. She stood behind me all the way e\ en while she taught me to think for myself and encouraged me to set ver\ high standards for myself. There was never a time when she was too busy to see me or critique my writing. She made available every opportunity to allow me to further my scientific career. She has been all that a student desires in a mentor and I am deeply appreciative of her professional support and kind friendship. I have been very fortunate to have an excellent committee. I would like to acknowledge members of my previous committee (Drs. Daniel Hardy, Daniel Webster, Harry Weitlauf, Laurel Donahue and Kurt Droms,) as well as my current committee (Drs. Beverly Chilton, Curt Pfarr , Daniel Webster, Sandra Whelly, Thomas Pressley and Vaughn Lee) for their invaluable technical insights and contributions to the development of this project and my own professional career. 1 owe my deepest appreciation to the scientific and technical input into this work from Dr. Aveline Hewetson and Ericka Hendrix for their substantial contributions to experimental design and troubleshooting. 1 am grateful to Dr. Peter Doris for his collaborative assistance with experiments discussed in Chapter V. I thank Brinda Dass, Oya Yazgan and Johnny Short for their advice and generous gift of reagents for the expression studies discussed in Chapter IV. This work was supported by NIH HD29457. Presentation of this work at various national and international meetings was also possible from funds from the same grant and tra\el mone> generouslv provided by the office of the Associate Dean for Research and the Graduate School, the Student Senate office at TTUHSC and a scholarship from the Helen Hodges Educational Charitable Trust. I thank all the members of the graduate school, including the graduate faculty, the students and the administrative staff, especially Alicia Call, Pain .lohnson and Pam Roddy for making my stay in this department and this institute a pleasant one. I ha\ e made several friendships here while strengthened others. I thank all my friends for their smiles and encouragement along the way. I especially thank Brinda Dass who is a gem of a person and who has helped me in my growth as a scientist and encouraged me to be my own person. To all my friends, with whom 1 shared many a glass of beer and enjoyed many a game of scrabble - THANK YOU !!!

Ill JABLE OF CONTEN IS

.'VCKNOWLEDGEMEN I S ii ABSTRACT vii LIST OF FIGURES ix LIST OF ABBREVIATIONS x CHAPTER I. INTRODUCTION 1 1.1 Uteroglobin 1 1.1.1 Uteroglobin Family of Proteins 2 1.1.2 Uteroglobin Function 3 1.2 Eukaryotic Transcription-A Review 4 1.2.1 Hormonal Regulation of Gene Expression 5 1.2.2 SWI/SNF Family of Chromatin Remodeling Proteins 8 1.3 Uteroglobin Promoter 9 1.4 RUSH Family of Proteins 11 1.5 RING Finger Motif 13 1.6 Purpose of Study 14 1.7 References 18 II. CLONING AND CHARACTERIZATION OF THE RING FINGER BINDING PROTEIN (RFBP) 28 2.1 Introduction 28 2.2 Materials and Methods 30 2.2.1 Reagents, Antibodies and Cells 30 2.2.2 Cloning of the RING Motif 31 2.2.3 Cloning of the RING Finger Binding Partner 31 2.2.4 Genomic Cloning 34 2.3 Results 35 2.3.1 Cloning of the RING Finger Binding Protein 35 2.3.2 ClustalW Alignment and Hydrophilicity Analysis of RFBP Identify it as a Type IV P-type ATPase 36

iv 2.3.3 RFBP Gene lacks Region D 38 2.4 Discussion 38 2.5 References 55 III. SUBCELLULAR LOCALIZATION OF RFBP 58 3.1 Introduction 58 3.2 Materials and Methods 59 3.2.1 Reagents and Antibodies 59 3.2.2 Subcellular Fractionation of Endometrial Tissue 60 3.2.3 Polyacrylamide Gel Electrophoresis and Western Blotting 60 3.2.4 Immuno Electron Microscopy 61 3.2.5 Animal Treatments 61 3.3 Results 62 3.4 Discussion 63 3.5 References 69 IV. IDENTIFICATION OF THE RUSH BINDING SITE IN RFBP 71 4.1 Introduction 71 4.2 Materials and Methods 73 4.2.1 Reagents and Antibodies 73 4.2.2 Preparation of Whole Cell and Nuclear Lysates 74 4.2.3 Synthesis of GST-RING Fusion Protein 74 4.2.4 Synthesis ofRFBP Fusion Protein 75 4.2.5 Co-immunoprecipitation and GST Pulldown using Cell Extracts or Recombinant RFBP 76 4.3 Results 77 4.4 Discussion 78 4.5 References 84 V. QUANTIFICATION OF RFBP IN ESTROUS AND HORMONALLY MANIPULATED ENDOMETRIUM USING COMPETITIVE QUANTITATIVE RT-PCR 87 5.1 Introduction 87 5.1.1 Hormonal Regulation of P-type ATPases 87 5.1,2 Competitive RF-PCR 88 5.2 Materials and Methods 89 5.2.1 Reagents 89 5.2.2 Animal Ireatments 90 5.2.3 Competitor Synthesis 90 5.2.4 Competitive RT-PCR 91 5.2.5 HPLC Analysis of Reaction Products 92 5.2.6 Data Analysis 92 5.3 Results 92 5.4 Discussion 94 5.5 References 101 VI. CONCLUSIONS AND DISCUSSION 102 6.1 Summary of Results 102 6.2 Discussion 105 6.2.1 Lamins 108 6.2.2 Lamin-binding Proteins 108 6.2.3 Actin 109 6.3 Future Studies 109 6.4 References Ill

VI ABSTRACT

Molecular regulation of Uteroglobin gene expression by progesterone and prolactin is mediated by RUSH transcription factors. The RUSH family of proteins, which includes rabbit RUSH-la and P and the human, mouse and plant homologs of RUSH la, are SWI/SNF related chromatin remodeling proteins. These proteins have a novel C3HC4 RING finger, at their -COOH terminus that has been implicated in mediating protein-protein interactions. When this motif was identified in RUSH proteins, it was used to screen an expression library to isolate cDNAs for proteins that complex with it. A single phage clone (~1.6kb insert) was identified. Sequence analysis of this RING Finger Binding Protein (RFBP) clone, revealed a partial cDNA that lacked an initiator codon but contained a stop codon. RACE PCR was then used to extend the 5' and 3' ends of the cDNA. The predicted amino acid sequence from the composite cDNA sequence (4286-bp) is that of a putative Type IV P-type ATPase. P-type ATPases are membrane transporters that use the energy of ATP hydrolysis to transport substrate across the membrane. Genomic cloning and ClustalW alignment indicate that RFBP is an atypical P-type ATPase that has only seven of eight core regions and nine of ten transmembrane domains typical of this family of proteins. Core region D that contains transmembrane domain four is absent from this protein. Western blot analysis, coupled with immunoelectron microscopy data, indicates that RFBP is present in the inner nuclear membrane. Coimmunoprecipitation and GST pulldown experiments showed a direct interaction between RUSH and RFBP. The RUSH binding site lies within aa 612 804 of the RFBP protein. Competitive quantitative RT-PCR indicates that RFBP is ubiquitous in its expression, with the expression pattern correlating with that of RUSH in these same tissues. In addition, expression of RFBP is hormonally regulated in the endometrium, suggesting that RFBP function and expression may be closely linked with the function of the RUSH proteins in regulating gene expression in the reproductive system. Current studies provide important information about RFBP as a RUSH binding partner. However further studies are required to further understand the mechanistic

vii interaction between the proteins to shed light on the mechanism of hormone regulation of uteroglobin gene expression.

Vlll LIST OF FIGURES

1.1 - Organization of the UG Gene 15 1.2 - Comparison of ATPase Motifs in HIPl 16, hHLTF-1, RUSH-la and p 16 1.3 - The RING Mofif 17 2.1 - Strategy for RACE PCR Reactions 41 -) -) - ClustalW Alignment ofRFBP with Type IV P-type ATPases 42 ~i •; - Hydrophilicity Profile 46 2.4 - Alignment of Nucleotide and Predicted Amino Acid Sequences for RFBP 47 2.5 - Characterization of PCR products with Electrophoresis and Sequence Analysis... 52 2.6- P-type ATPase Family 53 2.7 - Structure of a Classical P-type ATPase 54 3.1 - Western Analysis 65 3.2 - Possible Topology ofRFBP 66 3.3 - Immunolabeling of Ultra-thin Sections 67 3.4- Topology and Structure of RFBP 68 4.1 - Synthesis of GST-RING Fusion Protein and GST Pulldown Assay using Whole Tissue and Nuclear extracts 81 4.2- RUSH and RFBP Interact w vzvo 82 4.3 - Synthesis ofRFBP Fusion Protein followed by a GST Pulldown using Recombinant GST-RING Fusion Protein and in vitro Synthesized Recombinant RFBP 83 5.1- Outline for the Construction of Competitor RNA from a PCR MIMIC 96 5.2- Validation ofthe Competitive RT-PCR system 97 5.3 - Precision ofthe Single Tube Measurement 98 5.4- RFBP Expression is Hormone-dependent 99

IX LIST OF ABBREVIATIONS

.\P1.2 - Adaptor primer 1,2 ARP - Actin-related protein B.\F - Barrier to autointegration factor BAPl -BRC.Al associated protein-1 BARDl - BRCAl associated RING domain 1 BRC,\-1 - Breast/Ovarian cancer susceptibility gene-1 BRG1 - Brahma-related gene 1 brm brahma CCIO - Clara Cell lOkDa protein C/EBP - CCAAT/enhanver binding protein COOH-terminus - carboxyl terminal end Cys (C) - Cysteine GST - glutathione-S-transferase HHLTF - human helicase-like transcription factor His (H) - Histidine HP 1 - Heterochromatin protein 1 HPLC - High performance liquid chromatography HRE - Hormone response element INM - Inner nuclear membrane IPTG - Isopropyl-P-D-thiogalactopyranoside kDa kiloDalton KLH - Keyhole limpet hemocyanin L2BP1 - LAP2 binding protein 1 LAP 1,2 - Lamina associated polypeptide 1,2 LBR - Lamin B receptor MGB - Mammaglobin M/SARs - Matrix/Scaffold associated regions NGS - Normal goat serum NH^-terminus - amino terminal end OCT/Oet - Octamer P.\l-1 - Plasminogen activator inhibitor-1 PBS - Phosphate-buffered saline PMl. - Prom\ eloe\ tic leukemia oncoprotein POD - PML oncogenic domains PRL - Prolactin RACE - Rapid amplification of cDNA ends RFBP - RING finger binding protein RING - Realh Interesting New Gene RT-PCR - Reverse transcriptase-polymerase chain reaction SDS - Sodium dodecyl sulphate SERCA - Sarcoendoplasmic reticulum calcium ATP SIN - switch independent SNF - Sucrose nonfermenting sue - Sucrose SWI switch TAF - TBP associated proteins/factors TBP - TATA-binding protein TBS-T - Tris-buffered saline-tween TD - Transmembrane domain UG - Uteroglobin

XI CHAP I ER I INTRODUCTION

1.1 Utero^lobin From the moment a blastocyst is formed, its early development must be closely coordinated w ith the uterine environment to ensure its survival. There is a narrow window of time during which the endometrial epithelium is receptive to the embryo and experimental interference with the biochemical or endocrine uterine environment prevents successful implantation. Experiments using asynchronous egg transfer (Chang, 1950: No\ es et al.. 1963) showed that there was a need for strict synchronization ofthe embryonic and maternal systems indicating that there is specific biochemical cross-talk between the two. Depending upon the species, implantation may be either invasive, i.e., the embryo breaks through the surface epithelium to invade the underlying stromal tissues, or it may be non-invasive whereby the epithelial integrity is maintained during the implantation process. The human species, like the rabbit, mouse, dog and cat, exhibits the invasive mode of implantation. There is significant information available, for each of these species, on the suitable endocrine signals required for the generation of a congenial uterine environment resulting in the establishment ofthe secure physical anchorage ofthe blastocyst to the uterus. For several decades now, the rabbit has been commonly used as a model of choice, for the study of these processes, because ofthe close similarity with the process in humans. It has been useful in determining the biochemical basis ofthe critical molecular conversation that occurs during the stages of pregnancy starting with events during pre-implantation. It was back in 1965 that investigators first observed changes in protein patterns in uterine washings from pre-implantation stages in rabbits (Schwick, 1965). Then two investigators independently isolated a major component of these secretions and called it blastokinin (Krishnan and Daniel, 1967) and uteroglobin (Beier, 1968). An immunological response to anti-blastokinin serum was also seen in human samples (Johnson et al., 1972; Noske and Feigelson, 1976) indicating that this protein is also present during human pregnaiie>, Investigators then concentrated their efforts on characterizing this piDlein and its contribution to implantation. Biochemically, Uteroglobin or UG, as this protein has since been known, is a 16 kDa (Murra> ct al., 1972) homodimeric protein (McCiaughey and Murray, 1972) that is s\ nthesized by endometrial epithelium (Warembourg el al., 1986), Its expression is specificalK induced by progesterone (Beato, 1977) and it peaks at day 5 of pregnancy, dropping to basal le\el b\ day 10, Treatment of ovariectomized rabbits with pharmacological doses of progesterone for 5 days results in an increase in UG expression (Shen ct al.. 1983) whereas prolonged treatment (for 10 days) inhibits UG production due to an inhibitory effect of chronic progesterone treatment on the progesterone receptor concentration (Rahman et al., 1981), Estradiol alone for 5 days has no significant effect on the L'G content and, in fact, it has an anti-progestational action when given to progesterone stimulated rabbits (Lee and Dukelow, 1972), In the rabbit, UG has also been found in the oviduct, the male genital tract, the digesti\ e tract and the respiratory tract (Beier et al., 1975; Kay and Feigelson, 1972; Kirchner, 1976; Miele et al., 1987; Noske and Feigelson, 1976), However, the hormonal regulation of UG secretion varies in the different tissues. Whereas in the uterus, UG is induced by progesterone, its synthesis in the oviduct is under the control of estradiol (Goswami and Feigelson, 1974) and glucocorticoids regulate its expression in the lung.

1.1.1 Uteroglobin Family of Proteins Radioimmunoassays and fluorescence microscopy experiments conclusively proved the existence of UG in human reproductive tissues (Kikukawa et al., 1988; Manyak et al., 1988) and in human neonatal tracheobronchial washings (Dhanireddy et al., 1988). Jackson et al. (1988) also purified the protein from urine of patients with renal failure suggesting that the protein is present in the plasma or kidney. Advances in cloning technology allowed the isolation of a UG-like clone from a human lung cDNA expression library (Singh et al., 1988). This protein was called the Clara cell 10 kDa protein (CCIO) as it has an apparent molecular weight of 10 kDa in its nonreduced state. More recently, in the late 1990s, investigators have identified UG-like proteins in se\ eral tissues that are closely related to UG in their primary sequence. Mammaglobin (MGB) is present in human mammary epithelial cells (Watson and Fleming, 1996) and differentially expressed in several breast cancer cell lines while frequently over expressed in primary human breast tumors. A closely related protein, MGB2 (Becker et al, 1998) is expressed in neoplastic tissue. Lipophilins A, B and C form a family of proteins (Lehrer et al, 1998; Zhao et al., 1999) that are widely expressed in several tissues, especially endocrine responsix e tissues. These proteins are similar to a closely related protein called prostatein, a secreted protein ofthe rat prostate. Quite obviously, the roster ofthe uteroglobin gene superfamily members has increased and investigation into the properties of some of these proteins will further enhance the understanding of UG biology.

1,1.2 Uteroglobin Function The function of UG is still an enigma. While a great deal is now known about its regulation and structure, the presence of this protein in reproductive and non-reproductive tissues and its differential regulation in each of these tissues has confounded scientists for several decades. Its biochemical property of binding to progesterone was initially interpreted as being a clue to its function. It was proposed to be a progesterone carrier (Popp et al, 1978) and therefore involved in the maintenance of high intraluminal progesterone concentration in the pre-implantation uterus (Bochskanl et al., 1984). However this theory did not gain ground for lack oiin vivo evidence. It was then proposed that the protein had immunosuppressive and anti-inflammatory properties. Mukherjee et al. (1980, 1982) showed that UG cross-links with the transplantation antigens on the embryonic surface, thereby protecting it from the maternal immune system. This cross-linking is catalyzed by transglutaminase, a blood-clotting factor. UG also inhibits phospholipase A2 activity (Levin et al., 1986) thereby preventing the generation of potent lipid mediators of inflammation known to generate uterine smooth muscle contractility. Generation of UG knockout mice (Zhang et al., 1997b) by gene targeting in embryonic stem cells results in mice with severe fibronectin-deposit renal glomerular disease indicating a role for UG in preventing glomerular fibronectin deposits. An alternate technique for generating UG knockout mice (Stripp et al, 1996) however, resulted in mice that were unable to accumulate the environmental pollutants, poh chlorinated bipheiiyls, in their lungs but did not show the abnormal fibronectin deposit phenotype. Development of a transgenic mouse model expressing UG antisense mRNA (Zheng ct al., 1999) resulted in the generation of mice with suppressed UG production, that showed abnormal fibronectin and IgA deposition in the glomeruli. Recent studies have also implicated UG in reversing the transformed phenotype of cancer cells (Zhang ct al., 1999) suggesting tumor suppressor-like effects for the protein. A protein initial 1\ identified for its putative role in establishment and maintenance of pregnane) is now suggested to have a broader range of possible biological function, be\ ond e\'ents within the uterus during early pregnancy. The growing list of UG properties/functions has intrigued investigators for the past several years and laboratories around the world are investigating the multifiinctional nature of this protein. At the same time, the molecular events through which the tissue specific regulation of UG gene expression is mediated are only partly understood. Transcriptional regulation of gene expression is comprised of multiple, functionally distinct steps, including chromatin remodeling and transcriptional activation. Uteroglobin is therefore being used as a model for the study of tissue specific regulation of gene expression. It is anticipated that further study ofthe mechanism of its expression will \'ield significant insights into the finely regulated patterns of gene expression which coordinate processes involved in development, differentiation and reproduction.

1.2 Eukaryotic Transcription -A Review All eukaryotic protein-coding genes contain DNA sequence elements that are required for their regulation. These sequence elements fall into two classes, promoters and enhancers. The promoter region is located immediately upstream ofthe transcription start site and is required for accurate and efficient initiation of transcription (Dynan and Tjian, 1985). The typical promoter contains an AT-rich region, 20-30-bp upstream ofthe cap site, designated the TATA box. The TATA box binds the basal level transcription complex through the TATA-binding protein (TBP) and determines the direction and start site for transcription. A significant number of genes have also been identified that have a GC-rich region instead of a TATA box (Salbaum el a/,, 1988; Terao et al, 1983). The GC-rich stretch of 20-50 nucleotides occurs within the first 100-200 bases upstream of the start site and initiates transcription. Promoters are regulated by a combination of sequence-specific DN/\-binding proteins, general transcription initiation factors and associated accessor) factors. The enhancer sequence, on the other hand, is a c/.s-acting element distal to the transcription start site that works in an orientation-independent manner. These elements work to increase the rate of transcription from the promoter (Grosschedl and Birnstiel, 1980). When a 366-bp DNA segment ofthe SV40 promoter region was linked to a 3-globin gene, transcription ofthe gene was enhanced up to 200- fold (Banerji e/a/., 1981).

Enhancer motifs required for transcriptional induction for various genes have been identified. The transcription factors that recognize and bind these DNA elements ha\ e also been identified. For example, Davidson et al. (1986) conducted DNase I footprinting experiments to show that different sequence motifs within the SV40 enhancer are recognized by different factors present in HeLa and B cell nuclear extracts. Sequences specifically protected by HeLa cell factors are necessary for in vivo enhancer function in HeLa cells but not in B cells. Similarly, mutations in the regions protected only by B cell factors alter expression in B cells but not in HeLa cells. Whether or not a specific gene is expressed in a particular cell at a particular time is largely a consequence ofthe presence ofthe specific transcription factor in the cell and the accessibility of its binding site. A class of transcription factors that binds to enhancer elements includes the nuclear hormone receptors. These receptors modulate the expression of target genes in response to binding of hormone.

1.2.1 Hormonal Regulation of Gene Expression All vertebrates have effector molecules, called hormones, that are synthesized and secreted by one cell but elicit a response in another cell. The target cell may be either close to the site of secretion or further away in the body. Hormones are subdivided broadly into (a) hydrophilic peptides (FSH, LH, PRL, etc.) and amines (dopamine. noradrenaline and adrenaline) and (h) hydrophobic lipids that include eict)sanoids (prostaglandins) and steroids (progestagens, androgens, estrogens). Steroid hormones are all derived from a common precursor, cholesterol. Becau.se steroid hormones are lipid soluble, lhe\ can pass intact through the cell membrane ofthe target organ cells, as was demonstrated for aldosterone and estrogens by Porter el al. (1964) and Stumpf e/ al. (U)60), respectively. Once inside the cell, these steroids bind receptors (Bruchovsky and Wilson. l'-)68; Jensen and .laeobson, 1962). Characterization and purification of these hormone-binding proteins, coupled with the cloning of their cDNAs provided some insight into the molecular and functional architecture of steroid receptors. These hormone receptors are proteins composed of several domains which are differentially conserved between the various receptors and have different roles: a variable N-terminal region, a conserved DNA binding domain (DBD), a variable hinge region, a conserved ligand binding domain (LBD), and a variable C-terminal region. This C-terminal region specifies dimerization properties and contains a ligand-dependent transactivation function. As early as 1968 a two-step mechanism of action was proposed for these receptors based upon the observation of an inactive and an active state of the receptors (Fang et al., 1969; Jensen et al., 1968). The first step involves activation through binding ofthe hormone; the second step consists of transfer of this complex to the nucleus and receptor binding to DNA. Clever and Karlson (1960) had shown puff induction by ecdysone in giant chromosomes of insects indicating that steroids act at the genomic level in the cell. Gene transfer techniques allowed the identification of DNA sequences responsible for regulation by steroid hormones in the promoters of inducible genes (Yamamoto, 1985), These sequences called hormones response elements (HRE) are recognized by the ligand bound receptors. An HRE consists of two half-sites. The identity of a response element resides in three features: the sequence ofthe base pairs in the half-site, the number of base pairs between the half-sites and the relative orientation ofthe two half-sites. Thus each receptor protein dimer that binds the DNA has to recognize the sequence, spacing and orientation ofthe half-sites within their response element. However the nuclear hormone receptor does not function in isolation, it modulates gene expression by acting in concert with components of the transcription complex. Significant advances have been made in identifying the key players in this scenario and their modes of action/interaction. The caiboxyl terminal domain of nuclear hormone receptors not only determines the ligand binding and dimerization properties, but it also mediates the ligand-dependent transacti\ ation functions (Glass, 1994). These receptors exert their effect through cell t\ pe specific 1 .A FA-binding protein (TBP)-associated proteins (TAFs) and non-TAF proteins that physically and functionally interact with the liganded receptor (Cavailles et al., 1995; Halachmi ct al., 1994; Jacq et al., 1994). Another class of co-activators that ha\e been implicated in this role are the CBP (cAMP response element binding protein [CREB]-binding protein)/p300 family of proteins (Chakravarti et al., 1996; Kamei et al., 1996). These proteins contain histone acetyltransferase activity and can direct nucleosomal modification at the target promoters thereby affecting the accessibility of DN.A. for binding of regulatory proteins and transcription factors. DNA in the cell nucleus is organized into higher order chromatin structure. It is packaged into chromatin in orderly repeating protein-DNA complexes called nucleosomes. Each nucleosome consists of approximately 146-bp of double stranded DNA wrapped around a core of 8 histone proteins: 2 molecules each of H2A, H2B, H3 and H4 which form a histone core. Histone HI binds to the linker region between nucleosomes. Distortion ofthe periodicity ofthe superhelix and electrostatic shielding by the positively charged histones constrains the access of non-histone proteins to nucleosomal DNA. Therefore, for the template to become accessible to transcriptional machinery, remodeling of chromatin structure is required. This can occur by two processes, histone acetylation and/or ATPase-mediated DNA-histone dissociation. The former is a post-translational modification of histones by acetylation, resuhing in changes in electrostatic attraction for DNA and steric hindrance introduced by the hydrophobic acetyl groups which leads to destabilization ofthe interaction of histones with DNA. The second mechanism for alteration of chromatin is less well understood and involves ATP- dependent remodeling of chromatin to make it more accessible to transcription factors. 1.2.2 SWI/SNF Family of Chromatin Remodelina Proteins Genetic analysis in yeast has provided immense insight into the general in\ol\ement of chromatin structure in gene regulation. The product ofthe HO gene in Siiccluiromyccs ccrcvi.\iae is a site-specific endonuclease that initiates the mating type switching process. The >east Sll'Il, SWI2 and SWI3 genes were identified as positive regulators oi HO transcription (Stern el al., 1984) since mutations in these genes did not S\\ Itch on the expression of HO gene. Strains containing mutations in any of these genes exhibit a large number of defects similar to those due to a truncation ofthe carboxyl terminal domain of RNA polymerase II (Nonet et al., 1987) suggesting that SWIl, SWI2 and SW13 play a general role in transcription. The SWII, SWI2 and SWB genes are functionally similar to the SNF2, SNF5 and SNF6 genes iSNF for sucrose nonfermenting) that are required for transcription ofthe SUC2 gene (Peterson and Herskowitz, 1992). The SUC2 gene encodes invertase that is an enzyme required by S. cerevisiae to catabolize sucrose to raffinose. Genetic studies have identified the SNF genes as essential to this process. SWI2 and SNF2 are in fact identical (Laurent et al., 1991). Mutations in any of these genes result in a similar phenotype, suggesting that the genes code for members ofthe same protein complex. A cormection between the SWI/SNF complex and chromatin structure was first evidenced by the fact that transcriptional defects of swil', SM'i2' and/or swi3~ mutants are suppressed by mutant alleles of two genes, SIN] and SIN2 (SIN for switch independent). These two genes code for a high mobility group (HMG)-l like protein, that is a non-sequence-specific DNA binding protein, and histone H3. Moreover, the predicted SWI/SNF protein contains sequences typical ofthe consensus motifs found in helicases which are known to be involved in chromatin decondensation during replication (Gruss and Sogo, 1992; Laurent et al., 1992). The SWI/SNF protein also has double-stranded DNA-stimulated ATPase activity that is functionally related to the helicase activity of these proteins, further implicating these proteins in antagonizing the repressive effects of chromatin.

Soon after the discovery ofthe widespread involvement of SWI/SNF in transcriptional activation in yeast, a homolog of this protein was identified in Drosophila, called brahma ibrm', Tamkun et al., 1992). Brahma is required for the developmental control of .several ofthe homoeotic genes that define segmental identity in the Dro.sophila embr>o. The himian homolog of this protein, brahma-related gene 1 or BRGl was subsequently identified by Khavari et al. (1993) in a HeLa cell cDNA library. It was show n that a SIVI2/BRG1 chimera restored normal mitotic growth to poorly growing ,vu';'2" cells indicating functional conservation ofthe proteins between yeast and humans. The SW 1/SNF proteins form a stable complex that is approximately 2MDa in size (Wang et al., 1996). The purified complexes have intrinsic ATPase activity and function by coupling ATP hydrolysis to nucleosomal remodeling to facilitate the interaction of transcription factors with these promoters (Kwon et al., 1994). Ligand-dependent interactions ha\e been individually demonstrated between the glucocorticoid receptor and the estrogen receptor with the yeast SWI/SNF proteins (Yoshinaga et al., 1992) and also between the estrogen receptor and the human homologs of SWI2/SNF2 (Ichinose et al., 1997). The experimental results demonstrate that the yeast and human SWI/SNF proteins enhance transcriptional activation by the nuclear receptors resulting in ligand-dependent remodeling ofthe chromatin template of inducible genes. Several mechanistic studies suggest that SWI/SNF, and its related complexes in higher organisms, are targeted to specific regions ofthe genome by interactions with sequence-specific activators (Fryer and Archer, 1998; Ostlund Farrants et al., 1997)). The physical interaction between the glucocorticoid receptor and the BRGl complex was eliminated by the addition of an antiprogestin drug, which also blocked chromatin remodeling and transcriptional activation by the receptor on the target gene (Fryer and Archer, 1998).

1.3 Uteroglobin Promoter As mentioned earlier, UG is regulated differentially in the various tissues in which it is expressed. Extensive research is underway to identify the contribution of several transcription factors that have been implicated in regulating the expression of UG or its related, UG-like, proteins. DNase 1 protection assays, linker scanning analyses and promoter deletion studies earlier identified seven distinct regions, regions 1 through VII, centered at -30, -70, -95, -130, -190, -230, and -255, which contribute to the basal expression levels of UG in endometrial and lung epithelial cell lines (Misseyanni et al.. IWl; Suske cl al., 1992), As shown in Fig, 1.1, region VII is defined by the presence of an estrogen-response element (-265/-252). The ubiquitous octamer-binding protein OCT- 1 binds to regions III and IV; and Spl and Sp3 bind to regions II and VI. Region I contains a noncanonical TATA box (TACA box; Klug el al., 1994). Other regions ofthe promoter include a motif that is 92% identical to the GT-l motif in the Simian Virus 40 (SV40) enhancer (-258/-220), and a 7-bp identical inverted repeat (CAGTTTC) that is found in the long terminal repeat (-171/-148) of all murine leukemia viruses and proviruses (Wolfe/ al., 1992). Two HNF3-response elements centered at -130 and -95 complete!) overlap the OCT-1 binding sites in regions III and IV (Fig 1; Braun and Suske, 1998). These elements contribute to the activity ofthe promoter in H441 cells, a lung epithelial cell line, by enliancing Spl mediated activafion of UG/CCIO expression. Cassel et al. (2000) have also implicated proteins ofthe C/EBP family, C/EBPa and C/EBP5 in regulating differentiation-dependent expression ofthe CCIO gene. A noncanonical estrogen responsive element is located at a site 288-bp upstream ofthe transcription start site (Slater et al., 1990).

While Clara cells show constitutive expression of uteroglobin, which is modulated by glucocorticoids, expression in the endometrium is inducible by progesterone and estradiol. Evidence for the participation of chromatin structure in the regulated expression of the UG gene was initially derived from an analysis of DNasel hypersensitive sites in chromatin after hormone induction (Jantzen et al., 1987). Four DNasel hypersensifive regions were found at positions, -3.7, -2.4, -0.1 and +4.1-kb relative to the transcription start site ofthe UG gene. The three upstream sites are only detected in the hormonally stimulated endometrium while the +4.1 DNasel hypersensitive region is not tissue specific. Experiments with nuclei prepared from hormonally manipulated rabbits revealed that the region -2.4-kb upstream ofthe transcription start site is more susceptible to the presence of hormone than the -3.7 and -0.1 loci. Fine mapping of this region by DNA foot printing experiments revealed the presence of one strong progesterone binding site between -2.394 and -2.376 and two weaker binding sites between -2.426 and -2.402 and between -2.486 and -2.471-kb upstream ofthe transcription start site. There is also a cluster of three progesterone

10 receptor binding sites more than 100-bp upstream of this DNasel hypersensitive region extending from -2.709 to 2.627-kb upstream ofthe start site. It was already known by this time that progesterone affects the expression of UG messenger RN,\ in rabbit (Shen cl al, 1983) and human endometrium (Peri et al, 1994). This progesterone-induced effect in fact is further augmented by prolactin (Chilton and Daniel. U)S7; Daniel et al, U)S4). Treatment of long-term ovariectomized (>12 weeks) rabbits with either prolactin or progesterone results in a dramatic increase in the receptor for the other hormone (Chilton and Daniel, 1987; Chilton et al., 1988; Daniel el al, 1988; Randall et al. 1991). A servomechanism was proposed to explain the increase in endometrial UG mRNA, upon sequential treatment with prolactin plus progesterone, to a concentration equal to that found on the 5th day of pregnancy (Chilton et al, 1988). Gel shift assays with the UG promoter (Fig. 1.1; UG200; -194/+9) showed that sequences in the proximal part of the promoter are recognized by progesterone-dependent nuclear proteins (Rider and Bullock, 1988). Work by Kleis-SanFrancisco et al. (1993) showed that prolactin augments the binding of four progesterone-dependent proteins to an 85-bp 5'-flanking region (Fig. 1.1; UG99; -170/-85) ofthe UG gene. Recognition site screening finally allowed the isolation and cloning ofthe cDNAs for two of these proteins, RUSH- la and RUSH-ip, and identified them as new members ofthe SWI/SNF superfamily of nuclear receptor coactivators (Hayward-Lester et al, 1996). This further emphasizes the involvement of chromatin structure in regulating UG gene expression. A transient expression system with HRE-H9 uterine epithelial cells (Li et al, 1989) was then used to show that the proximal promoter region, that binds the RUSH proteins, is essential to the action of prolactin (Fig. 1.1; Hewetson and Chilton, 1997)

1.4 RUSH Family of Proteins The RUSH acronym identifies the key characteristics ofthe protein members of the family, i.e., RING finger motif protein cloned in rodent and rabbit, binds the Uteroglobin promoter, SWl/SNF-related, cloned in human, binds the HIV-1 promoter, and helicase-like. RUSH-la (1005-amino acids, 113 kDa) and RUSH-lp (836-amino acids, 95 kDa) bind the 85-bp (-170/-85) proximal promoter region ofthe rabbit

11 uteroglobin (UG) gene. RUSII-lot and p proteins are products of alternatively spliced messages and their a\ ailability is regulated by a steroid-dependent alternative splicing mechanism (Ilayward-Lester cl al, 1996), Competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and an ion-pair reversed phase high performance liquid chromatography (HPLC) product detection system showed that RUSH-la mRNA is the progesterone-dependent splice \ariant. The choice of acronym was thus influenced by the significance of alternative splicing as an adjunct to regulation ofthe uteroglobin promoter. When this posttranscriptional level of regulation is invoked, the response of the target cell to a physiological challenge is accelerated or RUSHed. Three independent teams of investigators have cloned the human homolog of RUSH-la by recognifion site screening of ^gt-11 cDNA expression libraries derived from HeLa cells. HIPl 16 (Sheridan et al, 1995), hHLTF (Ding et al, 1996), and Zbul (Gong et al, 1997) encode identical 1009-amino acid proteins (116 kDa). The human RUSH gene was mapped to a single locus on human chromosome 3q25.1-q26.1 (Lin et al. 1995), Activation ofthe plasminogen avtivator inhibitor-1 (PAl-l) gene in HeLa cells by hHLTF (Ding et al, 1996) and activation ofthe PAI-1 gene in 30A5 cells by PI 13 (the mouse homolog of RUSH-la [Zhang et al, 1997a]), provide in vivo evidence that RUSH proteins are transcription factors. The suggestion that RUSH proteins mediate the ability of prolactin to increase progesterone-dependent transcription ofthe uteroglobin gene (Hewetson and Chilton, 1997) is consistent with the hypothesis that RUSH proteins are targeted to an active promoter via an interaction with other transcriptional activators. All RUSH family members are characterized by the presence of seven consecutive domains that are typical of ATPases and DNA helicases (Fig. 1.2). Strong DNA- dependent ATPase acfivity has been demonstrated for HIPl 16, Zbul and PI 13, and supports the suggestion that RUSH proteins use the energy of ATP hydrolysis to disrupt chromafin structure. A key question is whether RUSH proteins associate with transcripfion factors, in a manner similar to the SWI/SNF proteins, to target RUSH proteins to specific genes (Devine et al, 1999). Gel shift, super-shift and immunodepletion assays were used to show that RUSH proteins bind to UG50 (Fig. 1.1; -160/-110), and UG50 competes for RUSH-specific

12 binding. C)clie Amplification and Selection of largets (CASTing; Pollock and Treisman, 1990) was further used to identify the putative RUSH target in the uteroglobin promoter (Chilton et al, 2000). Briefly, a hexameric core sequence of A/CCA/TTNG/T (MCWTNK) which is at times palindromic (ACATG 1) was identified after 5 cycles of selection. Preliminar) analysis of DNA-binding-site selectivity with gel shift assays indicated that the optimal consensus binding sequence is A/CCA/TTA/T/GG/T (MCWTDK), Although not previously identified as the binding site for any ofthe human (HIPl 16, hHLTF, Zbul) or mouse (PI 13) RUSH homologs, this consensus sequence is present in each ofthe probes used for library screens and/or gel mobility shift assays (Devine ero/„ 1999).

1.5 RING Finger Motif Sequence alignment of homologs from rabbit, human, rodent and plant (Devine et al, 1999) showed that all RUSH proteins contain the RING finger motif (C3HC4). The motif is a cy steine-rich amino acid sequence originally identified in the sequence ofthe Really Interesting New Gene I or RINGI. The motif binds two zinc ions in a unique cross- braced system (Fig. 1.3; Saurin et al, 1996) and is the common feature of a superfamily of nearly 200 otherwise unrelated proteins (Borden, 2000; Freemont, 1993). Many of these proteins are transcription factors, and some of them have oncogenic potential. Variants ofthe RING finger motif have also been found in several proteins. These include members ofthe RING-H2 family that have a His residue in place of Cys 4. Sometimes the mofif is present along with a B-box followed directly by a leucine coiled- coil thus forming a tripartite motif called the RBCC domain (Saurin et al, 1996). When the RING finger motif was first identified some thought it might play a role in DNA-binding because Zinc fingers are known as DNA-binding mofifs (Barlow et al, 1994; Freemont, 1993). However, in the absence of any data to support this idea, it is now believed that the RING motif mediates protein-protein interacfions (Borden and Freemont, 1996; Mackay and Crossley, 1998; Saurin et al, 1996). The search for functional binding partners ultimately led to the demonstration that the RING domain in one protein can associate with another RING domain to promote homo- or hetero-

13 dimerization. Alternatixel), the RING domain can also associate with non-RING domains (Borden, 2000). fhe recent discovery ofthe functional link between the ubiquitin-proteasome pathway and the RING containing protein Cbl has attracted considerable attention (.loazeiro cl al, 1999; Freemont, 2000). These studies support the idea that the RING motif is involved in ubiquitin-mediated proteolysis (Freemont, 2000); ho\\e\er Copps et al. (U)98) ha\e suggested that the RING motif may play a regulatory role in the ordered assembh of factors. When the RING domain was altered by site directed mutagenesis (Borden el al, 1995; Le et al, 1996), it was determined that the RING finger mofif of promyelocy tic leukemia oncoprotein (PML) is necessary for the formation of speckled nuclear bodies also known as promyelocytic oncogenic domains. More recently, Cao cl al. (1997) showed that mutations in the RING finger resulted in the retention ofthe ret finger protein in the cytoplasm even though the putafive nuclear localization signal was intact. The common underlying theme derived from all these studies is that the RING domain mediates protein-protein interactions giving rise to the speculation that the RING finger may, in fact, serve as a scaffold for the evolution of different functions (Borden, 2000).

1.6 Purpose of Study When we identified the RING finger motif in RUSH, our primary hypothesis was that the RUSH protein associated with another protein via the RING finger. This dissertation describes the cloning and characterization of this RING Finger Binding Protein (RFBP). In Chapter II, the cloning strategy for RFBP is described. In this chapter, I also discuss the identification ofRFBP as a putative Type IV P-type ATPase. Chapter III contains a description ofthe characterization ofthe subcellular distribufion ofRFBP protein in rabbit endometrial cells. In Chapter IV, I address the direct binding between RUSH and RFBP. In Chapter V, I describe the development of a competitive RT-PCR strategy that was ultimately used to determine the level ofRFBP message in different tissues in both male and female rabbits. The amount of message in hormonally manipulated female rabbits was also measured by this technique.

14 -2660 -2330 -395 4+9 BamH1 BamH1 I 1 ^ UG400 ^ ^ UG200

r UGQq -39? r^ifisiL

RNA :>-^

Figure 1.1. Organization ofthe UG Gene. The black boxes correspond to exons and introns are shown by open boxes. The 400-bp promoter region (-395/+9) is enlarged to show seven regions (I-VII) mapped by DNase I footprinting, deletion and linker scanning anahses. that contribute to the overall activity ofthe promoter. The positions of UG200 (-194/+9), UG99 (-170/-85) and UG50 (-160/-110) are shown. The progesterone response element is located 2.6-kb upstream ofthe transcription start site. RUSH binding site is located in the UG50 region upstream ofthe start site. TACA, TACA box; OCT, octamer factor binding sites; ERE, estrogen-responsive element.

15 la HIP, hHLTF 290 c; 11 AnnMHT (^Kn^TATAVI 478 LIICPLSVLSN IDQP3Q RUSH-la, [3 290 i;i r Anr>onKrLTAIAVI 475 LIICPLSVLSN IDQFt3Q

II HIP,hHLTF 546 LHSIKrtLRVILDEJGHAIRN 581 RWVLTGTPigSISLKDLWSLLSFL RUSH-la, |3 543 LHSIRWLRVILDECHAIRN 578 RWVLTGTPigSISLKDLWSLLSFL

III HIP,hHLTF 630 LRRLQSLIKNITLFiRTK 704 DVLGLLLRLRQICrUT RUSH-la,p 627 LRRLQSLIKNITLERRTK 701 DVLGLLLRLRQIOCHT

RING finger (CJHCL) HIP,hHLTF 760 CAICLDSLTVPVITHCAHVPCKPCICQ / IQNEQPHAKCPLCR RUSH-la,p 757 CAICXDSLTVPVITHCAHVFCKPCICQ cJlQNEQPHAKCPDCR

W HIP,hHLTF 853 KSLWSQFTTFLSLIE PLKASGFVFTRLDGSMAQKKR RUSH-la, 850 KSLWSQFTTFLSLIE h? PLKASGFVFTRLDGSMAQKKR

V VI HIP,hHLTF 909 LLSLKAGGVGaJSIL AASRVFLMDPAWNPAAEDQ FDRCHRLGQKQEVIITKFIVKDSVEENML RUSH-la, 906 LLSLKAGGVGLNL AASRVFLMDPASAJNPAAEDQ FDRCHRD3QKQEVIITKFIVKDSVEEM4L

Figure 1.2. Comparison of ATPase Motifs in HIPl 16, hHLTF-1, RUSH-la and p. ATPase motifs characteristic of ATPases and helicases are numbered 1 through VI. Boxed regions denote differences in amino-acids within the motifs. The presumptive nucleofide binding-motif in domain I is underlined and the highly conserved lysine (K), required for ATPase activity, is shown in bold.

16 ^9-39

Figure 1.3. The RING Motif. The domain called the RING finger or the C3HC4 finger is characterized by a cysteine rich sequence: Cys-X2-Cys-X(9.39)-Cys-X(i.3)-His-X(2-3)-Cys- X2-C\ s-X(4-48).Cys-X2-Cys. From the N-terminus, there are 3 cysteines, shown here in numbered ovals, followed by a histidine, labeled H, and then 4 cysteines again. Spacing between cysteines 1 & 2, 4 & 5 and between cysteines 6 & 7 is conserved. The RING finger binds two Zn2+ atoms (black ovals) per RING molecule and each atom is ligated tetrahedrally by either 4 cysteines or 3 cysteines and one histidine, in a unique cross- brace arrangement.

17 1.7 References Banerji, .1., Rusconi, S, and Schaffner, W (1981) I'xpression of a beta-globin gene is enhanced b) remote SV4() DNA sequences. Cell, 27, 299-308.

Barlow, P.N., Luisi, B,, Milner, A„ Elliott, M. and Everett, R. (1994) Structure ofthe C3HC4 domain by IH-nuclear magnetic resonance spectroscopy. A new structural class of zinc-finger. J Mol Biol, 237, 201-211.

Beato. M, (ed,) (1977) Hormonal control of uteroglobin synthesis. North Holland Publishing Co., North Holland, Amsterdam,

Becker, R.M,. Darrow, C, Zimonjic, D.B.. Popescu, N.C., Watson, M.A. and Fleming, T.P. (1998) Identification of mammaglobin B, a novel member ofthe uteroglobin gene famil), Genomics, 54, 70-78.

Beier, H.M. (1968) Uteroglobin: a hormone-sensifive endometrial protein involved in blastoe)st development. Biochim Biophys Acta, 160, 289-291.

Beier, H.M., Bohn, H. and MuUer, W. (1975) Uteroglobin-like anfigen in the male genital tract secretions. Cell Tissue Res, 165, 1-11.

Bochskanl. R.. Thie, M. and Kirchner, C. (1984) Progesterone dependent uptake of uteroglobin by rabbit endometrium. Histochemistry, 80, 581-589.

Borden, K.L. (2000) RING domains: master builders of molecular scaffolds? J Mo/ Biol, 295, 1103-1112,

Borden, K,L,. Boddy, M.N., Lally, J., O'Reilly, N.J., Martin, S., Howe, K., Solomon, E. and Freemont, P.S. (1995) The solution structure ofthe RING finger domain from the acute promyelocytic leukaemia proto-oncoprotein PML. Embo J, 14. 1532- 1541.

Borden, K.L. and Freemont, P.S. (1996) The RING finger domain: a recent example of a sequence-structure family. Cwr Opin Struct Biol, 6, 395-401.

Braun, H. and Suske, G. (1998) Combinatorial action of HNF3 and Sp family transcription factors in the activation ofthe rabbit uteroglobin/CC 10 promoter. J Biol Chem, 273, 9821-9S2S.

Bruchovsky, N. and Wilson, J.D. (1968) The intranuclear binding of testosterone and 5- alpha-androstan-17-beta- ol-3-one by rat prostate. J 5/o/ Chem, 243. 5953-5960. Cao, T., Borden, K.L., Freemont, P.S. and Etkin, L.D. (1997) Involvement ofthe rfp tripartite motif in protein-protein interactions and subcellular distribution. J C'e// Sci, 110, 1563-1571.

Cassel, T.N., Nordlund-Moller, L., Andersson, O., Gustafsson, J.A. and Nord, M. (2000) C/EBPalpha and C/EBPdelta activate the clara cell secretory protein gene through interaction with two adjacent C/EBP-binding sites. AmJRcspir Cell Mol Biol, 22, 469-480.

Ca\allies, V., Dau\ois. S., L'Horset, F., Lopez, G., Hoare, S„ Kushner, P.J. and Parker, M.G. (U)^)5) Nuclear factor R1P140 modulates transcripfional acfivation by the estrogen receptor. Emho J, 14, 3741-3751.

Chakravarti, D., LaMorte, V.J., Nelson, M.C., Nakajima, T„ Schulman, I.G,, Juguilon, H„ Montminy, M, and Evans, R.M. (1996) Role of CBP/P300 in nuclear receptor signalling [see comments]. Nature, 383, 99-103.

Chang, M,C. (1950) Development and fate of transferred rabbit ova or blastocysts in relation to the ovulation fime of recipients. J. Exp. Zoology, 114, 197-225.

Chilton, B.S. and Daniel, J.C, Jr. (1987) Differences in the rabbit uterine response to progesterone as influenced by growth hormone or prolactin. J Reprod Fertil, 79, 581-587.

Chilton, B.S., Hewetson, A., Devine, J., Hendrix, E. and Mansharamani, M. (2000) Uteroglobin Gene Transcription, What's the RUSH? Ann N Y Acad Sci, 923, 166- 180.

Chilton, B.S., Mani, S.K. and Bullock, D.W. (1988) Servomechanism of prolactin and progesterone in regulating uterine gene expression. Mol Endocrinol, 2, 1169- 1175.

Clever. U. and Karlson, P. (1960) Reduktion von Puff-Veranderungen in den Speicheldrusenchromosomen von Chironomus tentans durch Ecdyson. Exp Cell Res, 20, 623-626.

Copps, K., Richman, R.. Lyman, L.M., Chang, K.A., Rampersad-Ammons, J. and Kuroda, M.I. (1998) Complex formafion by the Drosophila MSL proteins: role of the MSL2 RING finger in protein complex assembly. Embo J, 17, 5409-5417.

Daniel, J.C, Jr., Jetton, A.E. and Chilton, B.S. (1984) Prolactin as a factor in the uterine response to progesterone in rabbits. J Reprod Fertil, 72, 443-452.

19 Daniel, J.C., .Ir., Juneja, S.C, Taylor, S.P., Lonergan, P.B., Sullivan, P.K. and Chilton, B.S. (1988) Variability in the response ofthe rabbit uterus to progesterone as influenced b) prolactin../ Reprod Fertil, 84, 13-21.

Da\ idson, I., Fromental, C, Augereau, P., Wildeman, A., Zenke, M. and Chambon, P. (1^)86) Cell-type specific protein binding to the enhancer of simian virus 40 in nuclear extracts. Xaliire, 323, 544-548.

Devine, J.ll,, Hewetson, A., Lee, V.H. and Chilton, B.S. (1999) After chromatin is SWltched-on can it be RUSHed',' Mol ('ell Endocrinol, 151, 49-56.

Dhanireddy, R., Kikukawa, T. and Mukherjee, A.B. (1988) Detection of a rabbit uteroglobin-like protein in human neonatal tracheobronchial washings. Biochem Biophys Res Comnnm, 152, 1447-1454.

Ding, H., Descheemaeker, K., Marynen, P., Nelles, L., Carvalho, T., Carmo-Fonseca, M.. Collen, D. and Belayew, A. (1996) Characterization of a hehcase-like transcription factor involved in the expression ofthe human plasminogen activator inhibitor-1 gene. DNA Cell Biol, 15, 429-442.

Dynan, W.S. and Tjian, R. (1985) Control of eukaryotic messenger RNA synthesis by sequence-specific DNA- binding proteins. Nature, 316, 774-778.

Fang, S., Anderson, K.M. and Liao, S. (1969) Receptor proteins for androgens. On the role of specific proteins in selective retention of 17-beta-hydroxy-5-alpha- androstan-3-one by rat ventral prostate in vivo and in vitro. J Biol Chem, 244. 6584-6595.

Freemont, P.S. (1993) The RING finger. A novel protein sequence motif related to the zinc finger. Ann N YAcad Sci, 684, 174-192.

Freemont, P.S. (2000) RING for destruction? Curr Biol, 10, R84-87.

Fryer, C.J. and Archer, T.K. (1998) Chromafin remodelling by the glucocorticoid receptor requires the BRGl complex. Nature, 393, 88-91.

Glass, C.K. (1994) Differenfial recognition of target genes by nuclear receptor monomers, dimers, and heterodimers. Endocr Rev, 15, 391-407.

Gong, X., Kaushal, S., Ceccarelli, E., Bogdanova, N.. Neville, C, Nguyen, T„ Clark, H., Khatib, Z.A., Valentine, M., Look, A.T. and Rosenthal, N. (1997) Developmental regulation of Zbul, a DNA-binding member ofthe SWI2/SNF2 family. Dev Biol, 183, 166-182.

20 Goswami, .\. and feigelson, M, (1^)74) Differential regulation of a low-molecular-weight protein in (w iductal and uterine fluids by ovarian hormones. Endocrinology, 95, 669-675,

Grosschedl, R. and Birnstiel, M.L. (1980) Spacer DNA sequences upstream ofthe T-A- T-,\-.\-.'\-f-.\ sequence are essential for promotion of H2A histone gene transcription in \ i\o, Proc Natl .leadSci US.4, 77, 7102-7106.

Gruss, C. and Sogo. J.M, (1992) Chromatin replication, Bioessuys, 14, 1-8.

Halachmi, S,. Marden, E,, Martin, G., MacKay, H., Abbondanza, C. and Brown, M. (1994) Estrogen receptor-associated proteins: possible mediators of hormone- induced transcription. Science, 264, 1455-1458.

Hay ward-Lester, A., Hewetson, A., Beale, E.G., Oefner, P.J., Doris, P.A. and Chilton, B.S, (1996) Cloning, characterization, and steroid-dependent posttranscriptional processing of RUSH-1 alpha and beta, two uteroglobin promoter-binding proteins, Mol Endocrinol 10, 1335-1349,

Hewetson, A. and Chilton, B.S. (1997) Novel elements in the uteroglobin promoter are a functional target for prolactin signaling. Mol Cell Endocrinol, 136, 1-6.

Ichinose, H.. Gamier, J.M., Chambon, P. and Losson, R. (1997) Ligand-dependent interaction between the estrogen receptor and the human homologues of SWI2/SNF2. Gene, 188, 95-100.

Jackson, P.J., Turner, R., Keen, J.N., Brooksbank, R.A. and Cooper, E.H. (1988) Purification and partial amino acid sequence of human urine protein 1. Evidence for homology with rabbit uteroglobin. J Chromatogr, 452. 359-367.

Jacq, X., Brou, C, Lutz, Y., Davidson, I., Chambon, P. and Tora, L. (1994) Human TAFII30 is present in a distinct TFllD complex and is required for transcriptional activation by the estrogen receptor. Cell, 79, 107-117.

Jantzen, K., Fritton, H.P., Igo-Kemenes, T., Espel, E., Janich, S., Cato, A.C., Mugele, K. and Beato, M. (1987) Partial overlapping of binding sequences for steroid hormone receptors and DNasel hypersensitive sites in the rabbit uteroglobin gene region. Nucleic Acids Res, 15, 4535-4552.

Jensen, E.V. and Jacobson, H.l. (1962) Basic guides to the mechanism of Estrogen action. In Pincus, G. (ed.) Recent Progress in Hormone Research. Academic Press, New York, Vol. XVIII, pp. 387-414.

21 Jensen, E.V., Suzuki, 1., Kawashima, P., Stumpf, W.i;,, Jungblut, P.W. and DeSombre, E.R. (1968) A two-step mechanism for the interaction of estradiol with rat uterus. Proc Will.4cadSci US.4, 59, 632-638.

Joazeiro, C.A., Wing. S,S„ Huang, H„ Leverson, J.D„ Hunter, T, and Liu, Y,C, (1999) The tyrosine kinase negative regulator c-Cbl as a RlNG-type, E2- dependent ubiquitin-protein ligase [see comments]. Science, 286, 309-312.

.lohnson. Mil, Cowan, B,D, and Daniel, J,C., Jr, (1972) An immunologic assay for blastokinin, Fertil Sleril, 23, 93-100.

Kamei. Y.. Xu. L,, Heinzel, T,, Torchia, J,, Kurokawa, R., Gloss, B,, Lin, S.C, Heyman, R.A„ Rose, D,W., Glass, C.K. and Rosenfeld, M.G. (1996) A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell, 85, 403-414.

Kay, E. and Feigelson, M. (1972) An estrogen modulated protein in rabbit oviducal fluid. Biochim Biophys .4cla, 271, 436-441.

Khavari. P.A., Peterson, C.L., Tamkun, J.W., Mendel, D.B. and Crabtree, G.R. (1993) BRGl contains a conserved domain ofthe SWI2/SNF2 family necessary for normal mitofic growth and transcription. Nature, 366, 170-174.

Kikukawa, T., Cowan, B.D., Tejada, R.I. and Mukherjee, A.B. (1988) Partial characterization of a uteroglobin-like protein in the human uterus and its temporal relationship to prostaglandin levels in this organ. J Clin Endocrinol Metab, 67, 315-321.

Kirchner, C (1976) Uteroglobin in the rabbit. I. Intracellular localization in the oviduct, uterus, and preimplantation blastocyst. Cell Tissue Res, 170, 415-424,

Kleis-SanFrancisco, S,. Hewetson, A. and Chilton, B.S. (1993) Prolactin augments progesterone-dependent uteroglobin gene expression by modulating promoter- binding proteins. Mol Endocrinol, 7, 214-223.

Klug, J., Knapp, S., Castro, I. and Beato, M. (1994) Two disfinct factors bind to the rabbit uteroglobin TATA-box region and are required for efficient transcripfion. Mol Cell Biol, 14,620S-6218.

Krishnan, R.S. and Daniel, J.C, Jr. (1967) "Blastokinin": inducer and regulator of blastocyst development in the rabbit uterus. Science, 158, 490-492.

Kwon, H., Imbalzano, A.N., Khavari, P.A., Kingston, R.E. and Green, M.R. (1994) Nucleosome disruption and enhancement of activator binding by a human SWI/SNF complex [see comments]. Nature, 370, 477-481.

09 Laurent, B.C., Freitel, M.A. and Carlson, M. (1991) Functional interdependence ofthe ) east SNl'2, SNF5, and SNF6 proteins in transcriptional activation. Proc Natl .4cad.Sci r\.l, 88, 2687-2691.

Laurent, B.C., ^'ang, X. and CarlscMi, M. (1992) An essential Saccharomyces cerevisiae gene homologous to SNI'2 encodes a helicase-related protein in a new family. Mol Cell Biol, 12, 1893-1902.

Le, X.F., Yang, P. and Chang, K.S. (1996) Analysis ofthe growth and transformation suppressor domains of promyelocytic leukemia gene, PML, J Biol Chem, 271, 130-135,

Lee. .\.E, and Dukelow, W,R, (1972) Synthesis of DNA and mitosis in rabbit uteri after oestrogen and progesterone injections, and during early pregnancy. J Reprod Fertil, 31, 473-476.

Lehrer, R.I,. Xu. G,, Abduragimov, A., Dinh, N.N., Qu, X.D., Martin, D. and Glasgow, B.J. (1998) Lipophilin, a novel heterodimeric protein of human tears. FEBS Lett, 432, 163-167.

Levin, S.W., Bufier, J.D., Schumacher, U.K., Wightman, P.D. and Mukherjee, A.B. (1986) Uteroglobin inhibits phospholipase A2 activity. Life Sci, 38, 1813-1819.

Li, W.I., Chen, CL. and Chou, J.Y. (1989) Characterization of a temperature-sensifive beta-endorphin-secreting transformed endometrial cell line. Endocrinology, 125, 2862-2867.

Lin, Y.. Sheridan, P.L., Jones, K.A. and Evans, G.A. (1995) The HIPl 16 SNF2/SW12- related transcription factor gene (SNF2L3) is located on human chromosome 3q25,l-q26.1. Genomics, 27, 381-382.

Mackay, J.P. and Crossley, M. (1998) Zinc fingers are sticking together. Trends Biochem Sci, 23, 1-4.

Manyak, M.J.. Kikukawa, T. and Mukherjee, A.B. (1988) Expression of a uteroglobin- like protein in human prostate. J Urol, 140, 176-182.

McGaughey. R.W. and Murray, F.A. (1972) Properties of blastokinin: amino acid composifion, evidence for subunits, and estimation of isoelectric point. Fertil Steril, 23, 399-404.

Miele, L.. Cordella-Miele, E. and Mukherjee, A.B. (1987) Uteroglobin: structure, molecular biology, and new perspectives on its function as a phospholipase A2 inhibitor. Endocr Rev, 8, 474-490.

23 Mis.seyanni, A,, Klug,.!.. Suske. G. and Beato, M. (1991) Novel upstream elements and the r.\ lA-box region mediate preferential transcription from the uteroglobin promoter in endometrial cells. Nucleic .4ciJs Res, 19, 2849-2859,

Mukherjee, A,B„ Laki, K, and Agrawal, A.K. (1980) Possible mechanism of success of an allotransplantation in nature: mammalian pregnancy. Med Hypotheses, 6, 1043-1055

Mukherjee, A.B., Ulane, R.E. and Agrawal, A.K. (1982) Role of uteroglobin and transglutaminase in masking the antigenicity of implanting rabbit embryos, ylw J Reprod Immunol, 2, 135-141.

Murray, F.A,. McGaughey, R.W. and Yarns, M.J. (1972) Blastokinin: its size and shape, and an indication ofthe existence of subunits. Fertil Steril, 23, 69-77.

Nonet, M., Sweetser, D. and Young, R.A. (1987) Functional redundancy and structural polymorphism in the large subunit of RNA polymerase II. Cell, 50, 909-915.

Noske, I.G. and Feigelson, M. (1976) Immunological evidence of uteroglobin (blastokinin) in the male reproductive tract and in nonreproductive ductal tissues and their secretions. Biol Reprod, 15, 704-713.

Noyes. R.W., Dickman, Z., Doyle, L.L. and Gates, A.H. (1963) Ovum transfers, synchronous and asynchronous, in the study of implantation. In Enders, A.C (ed.) Delayed Implantation. University of Chicago Press, pp. 197-211.

Ostlund Farrants, A.K., Blomquist, P., Kwon, H. and Wrange, O. (1997) Glucocorticoid receptor-glucocorticoid response element binding stimulates nucleosome disruption by the SWI/SNF complex. Mol Cell Biol, 17, 895-905.

Peri, A., Cowan, B.D., Bhartiya, D., Miele, L., Nieman, L.K., Nwaeze, I.O. and Mukherjee, A.B. (1994) Expression of Clara cell 10-kD gene in the human endometrium and its relationship to ovarian menstrual cycle. DNA Cell Biol, 13, 495-503.

Peterson, CL. and Herskowitz, I. (1992) Characterization ofthe yeast SWIl, SWI2, and SWI3 genes, which encode a global activator of transcription. Cell 68, 573-583.

Pollock, R. and Treisman, R. (1990) A sensitive method for the determination of protein- DNA binding specificities. Nucleic Acids Res, 18, 6197-6204.

Popp, R.A., Foresman, K.R., Wise, L.D. and Daniel, J.C, Jr. (1978) Amino acid sequence of a progesterone-binding protein. Proc Natl Acad Sci US A. 75, 5516- 5519.

24 Porter, G..\., Bogoroch, R, and Edclman, I.S, (1064) On the mechanism of action of .Mdosteione on Sodium transport: the role of RNA synthesis. Proc Natl Acad Sci US A, 52. 1326-1333.

Ralmian, S,S., Billiar, R.B. and Little, B. (1981) Studies ofthe decline of uteroglobin synthesis and secretion in the rabbit uterus during the continued presence of circulating progesterone. Endocrinology, 108, 2222-2227,

Randall, Ci W., Daniel, J,C., Jr, and Chilton, B,S. (1991) Prolactin enhances uteroglobin gene expression by uteri of immature rabbits. J Reprod Ferlil, 91, 249-257,

Rider, V. and Bullock, D.W. (1988) Progesterone-dependent binding of a trans-acting factor to the uteroglobin promoter. Biochem Biophys Res Commun, 156, 1368- 1375

Salbaum, J.M,. Weidemann, A., Lemaire, H.G., Masters, CL. and Beyreuther, K. (1988) The promoter of Alzheimer's disease amyloid A4 precursor gene. Embo J, 7, 2807-2813.

Saurin, A.J., Borden, K.L., Boddy, M.N. and Freemont, P.S. (1996) Does this have a familiar RING? Trends Biochem Sci, 21, 208-214.

Schwick, H.G. (1965) Chemisch-entwicklungsphysiologische beziehungen von uterus zu blastocyste des kaninchens (Oryctolagus cuniculus). Wilhelm Roux Arch. Etnw Meek Org, 156,283-343.

Shen, X.Z.. Tsai, M.J., Bullock, D.W. and Woo, S.L. (1983) Hormonal regulation of rabbit uteroglobin gene transcription. Endocrinology, 112, 871-876.

Sheridan, P.L., Schorpp, M., Voz, M.L. and Jones, K.A. (1995) Cloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs ofthe SV40 enhancer and to the HIV-1 promoter. J Biol Chem, 270, 4575-4587.

Singh, G., Katyal, S.L., Brown, W.E., Phillips, S., Kennedy, A.L., Anthony, J. and Squeglia, N. (1988) Amino-acid and cDNA nucleotide sequences of human Clara cell 10 kDa protein [published erratum appears in Biochim Biophys Acta 1989 Mar 1;1007(2):243]. Biochim Biophys Acta, 950, 329-337.

Slater, E.P., Redeuihl, G., Theis, K., Suske, G. and Beato, M. (1990) The uteroglobin promoter contains a noncanonical estrogen responsive element. Mol Endocrinol, 4,604-610.

Stem, M., Jensen, R. and Herskowitz, I. (1984) Five SWI genes are required for expression ofthe HO gene in yeast. J Mol Biol, 178, 853-868.

25 Stripp, B.R., Lund, J.. Mango, G.W., Doyen, K.C, .lohnston, C, Hultenby, K., Nord, M. and Whitsett, J.A. (1996) Clara cell secretory protein: a determinant of PCB bioaccumulation in mammals. Am J Physiol, 271, L656-664,

Stumpf, W,E, and Roth, L,.I, (1966) High resolution autoradiography with dry mounted, freeze-dried frozen sections. Comparative study of six methods using two diffusible compounds 3H-estradiol and 311-mesobilirubinogen. J//z,ytoc/zew Cvlochcni, 14,274-287.

Suske. G., Lorenz, W., Klug, J., Gazdar, A.f and Beato, M. (1992) Elements ofthe rabbit uteroglobin promoter mediating its transcription in epithelial cells from the endometrium and lung. Gene Expr, 2, 339-352,

Tamkun, J,\\ ,, Deuring, R,, Scott, M,P,. Kissinger, M., Pattatucci, A.M., Kaufman, T.C and Kennison, J.A. (1992) brahma: a regulator of Drosophila homeotic genes stmcturall) related to the yeast transcriptional activator SNF2/SW12. Cell, 68, 561-572.

Terao, M., Watanabe, Y., Mishina, M. and Numa, S. (1983) Sequence requirement for transcription in vivo ofthe human preproenkephalin A gene. Embo J, 2, 2223- 2228

U'ang, W.. Cote, J., Xue. Y., Zhou, S., Khavari, P.A., Biggar, S.R., Muchardt, C, Kalpana, G.V., Goff, S.P., Yaniv, M., Workman, J.L. and Crabtree, G.R. (1996) Purification and biochemical heterogeneity ofthe mammalian SWI-SNF complex. Embo J, 15, 5370-5382.

Warembourg, M., Tranchant, O., Atger, M. and Milgrom, E. (1986) Uteroglobin messenger ribonucleic acid: localization in rabbit utems and lung by in situ hybridization. Endocrinology, 119, 1632-1640.

Watson, M.A. and Fleming, T.P (1996) Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer. Cancer Res, 56, 860-865.

Wolf, M., Klug, J., Hackenberg, R., Gessler, M., Grzeschik, K.H.. Beato, M. and Suske, G. (1992) Human CCIO, the homologue of rabbit uteroglobin: genomic cloning, chromosomal localizafion and expression in endometrial cell lines. Hum Mol Genet, 1, 371-378.

Yamamoto, K.R. (1985) Steroid receptor regulated transcription of specific genes and gene networks. Annu Rev Genet, 19, 209-252.

26 \'oshinaga, S.K , Peterson, CL,, Herskowitz, I. and Yamamoto, K.R. (1992) Roles of SWIl, SW12, and SWI3 proteins for transcriptional enhancement by steroid receptors. Science, 258, 1598-1604.

Zhang, Q., Ekhterae, D. and Kim, K.H. (1997a) Molecular cloning and characterization of PI 13, a mouse SNF2/SW12- related transcription factor. Gene, 202, 31-37.

Zhang, Z., Kundu, G.C, Panda. D,, Mandal, A,K., Mantile-Selvaggi, G., Peri, A., Yuan, CJ, and Mukherjee, A,B, (1999) Loss of transformed phenotype in cancer cells h\ overexpression ofthe uteroglobin gene, Proc Natl Acad Sci U S A,96, 3963- 3968.

Zhang, Z., Kundu, G.C. Yuan, C.J., Ward, J.M., Lee, E.J., DeMayo, F., Westphal, H. and Mukherjee. A.B. (1997b) Severe fibronectin-deposit renal glomerular disease in mice lacking uteroglobin. Science, 276, 1408-1412.

Zhao, C. Nguyen, T., Yusifov, T., Glasgow, B.J. and Lehrer, R.I. (1999) Lipophilins: human peptides homologous to rat prostatein. Biochem Biophys Res Commun, 256, 147-155.

Zheng, F., Kundu, G.C, Zhang, Z., Ward, J., DeMayo, F. and Mukherjee, A.B. (1999) Uteroglobin is essential in preventing immunoglobulin A nephropathy in mice. .\atMed.5, 1018-1025.

27 ClIAPlERIl CLONING AND CHARACTERIZATION OF THE

RING FING1:R BINDING PROTEIN (RFBP)

2.1 Introduction The RING finger protein sequence was first identified nearly a decade ago, in the protein product ofthe human gene Rl\'Glor Really [nleresling New Gene 1, which was isolated b) \ iilue of its location proximal to the major histocompafibility region on chromosome 6. The conserved C3HC4 structure, characteristic of this motif, coordinates the binding of two zinc atoms (Freemont, 1993) in a unique cross-brace arrangement (Fig. 1,3 in Chapter I). As mentioned earlier, this arrangement allows for the first and third pair of zinc ligands, forming site 1, to share zinc 1 while the second and fourth pair of zinc ligands form site 2 and bind zinc 2. Site 1 has a higher affinity for zinc than site 2. This may allow the RING to bind one set of protein partners through site 1 and another, with different affinity, through site 2, The number of proteins known to have the RING domain has grown enormously and the motif and its variants have so far been identified in over 200 proteins that are diverse both in function and origin. Previously identified RING finger containing proteins participated in gene expression, DNA recombination and DNA repair. This observation coupled with the subsequent preliminary gel- retardation and surface charge studies implicated this motif in DNA binding (Lovering et al, 1993). However, evidence accumulated over the years has shown that unlike the Zinc finger domain, the RING motif is not just restricted to proteins present in the nucleus but is in fact found in several cytoplasmic proteins as well. Besides, the DNA-binding results can be attributed to the positive surface charge potential that can mediate non-specific binding to DNA as a result of charge-charge interactions. Notably, RING containing proteins are known to function in several cellular processes including transcription, RNA processing, organelle transport, peroxisomal biogenesis and ubiquitination, to name just a few. The unifying theme in these diverse processes is the ability ofthe RING to mediate protein-protein interactions; particularly in the formation of macromolecular scaffolds.

28 Probabl) one ofthe most famous RING finger containing protein is the product of the Breast C)\arian Cancer Susceptibility Gene, BRCAl, which functions as a tumor suppressor in the cell. The BRCAl RlN(i finger encompasses residues 1-64 at the N- terminus ofthe protein. That the RING is essential to BRCAl function is apparent from the fact that the RING motif is the most highly conserved motif among the BRCAl genes from human, mouse and rat (Abel el al, 1995) and several cancer-predisposing mutations are found in this region ofthe protein (Abel el al, 1995). Three proteins have been identified by )east two-hybrid analysis which interact with the RING domain of BRCAl (Houvras et al, 2000; Jensen el al, 1998; Wu et al, 1996). In addifion, the first 110 residues ofthe protein, that include the RING domain, form a stmctural domain that readil) homodimerizes in solution. Although the importance of BRCAl in tumor suppression and cell growth regulation is widely recognized, little is known about the fimctional significance of its homodimerization and its contribution to protein-protein interactions. Along those same lines, while several BRCAl RING-interacting proteins have been identified, investigations are under way to define the functional significance of these interactions. Similar strategies have however been commonly used to identify a binding partner for other RING finger containing proteins; for instance, in the identification of a binding partner for the RING finger domain of COP 1, a photomorphogenic protein of Arabidopsis. Its interacting partner is called CIP8 for COPl interacting protein (Torii et al, 1999). In all instances, the RING finger has been essential to the interaction and mutations or deletions in its sequence abolish protein-protein interactions. All RUSH protein family members contain the RING motif at their carboxy terminus. This sequence, CAICLDSLTVPVITHCAHVFCKPCICQVIQNEGPHAKCPLC is in good agreement with the consensus C3HC4 motif. The RING motifs, in different proteins, are significantiy diverse in terms of amino acid sequence and spacing between the zinc ligands. Because of this extreme variability, the domain can probably function with altered specificity in the different proteins. With all the evidence implicating the RING finger in mediating protein-protein interactions, when we identified the RING domain in RUSH proteins, we h\ pothesized that the RING finger motif in RUSII probably mediates the binding between RUSH and another as yet unidentified partner. We set out to identify a RUSH-RING finger binding protein using an expression strategy that is less laborious than the yeast two-hybrid technique and has been employed to isolate binding partners for the Jun protein (Maegregor et al, 1990). A RUSH-RING finger binding protein was indeed identified and called the RING Finger Binding Protein (RFBP), This chapter describes the isolafion and subsequent characterization ofthe cDNA for RFBP, A computer search of known sequence databases (SWISS-PROT + PIR + PRE and GenBank) using the BLAST 2.0.12 program at the National Center for Biotechnology Information was used to identify sequence identity/similarity with related proteins. Hydrophilicity analysis and ClustalW alignment were also carried out to further characterize the predicted primary sequence of RFBP.

2.2 Materials and Methods 2.2.1 Reagents, Antibodies and Cells cDNA Synthesis System Plus, cDNA Cloning System-iVgtll, Sequenase Version 2.0 DNA Sequencing Kit, [a-^"^S]dATP, 1000 Ci/mmol, for sequencing, and isopropyl-P- D-thiogalactopyranoside (IPTG) were purchased from Amersham Pharmacia Biotech (Piscataway, New Jersey). The SMART RACE and Marathon^'^^cDNA Amplification Kits, Rabbit Genomic Library, TaqStart antibody, Tth Start antibody. Advantage Genomic PCR kit, and Advantage Tth polymerase mix were purchased from Clontech Laboratories, Inc. (Palo Aho, CA). TaKaRa ExTaq enzyme and lOX LA PCR buffer were purchased from Pan Vera Corporation (Madison, WI). The vector pSCREEN-lb(+), Thrombin Cleavage Capture Kit, T7-Tag antibody alkaline phosphatase conjugate and S- protein alkaline phosphatase conjugate were purchased from Novagen (Madison, Wl). The pCR™II and pCR(DII-TOPO vectors are components of individual TA Cloning Kits that were purchased from Invitrogen (San Diego, CA). MetaPhor agarose was purchased from BioWhittaker Molecular Applications, Inc. (Rockland, ME). A Ikb DNA ladder was purchased from Promega (Madison, WI).

30 2.2.2 Cloning ofthe RING Motif For the purpose of cloning, the RING motif was amplified from 50 ng of a 1030- bp partial RUSH cDNA clone (Hayward-Lester el al, 1996). The 50 pi PCR reaction mix contained LA PCR buffer (IX), I'aKaRa ExTaq DNA polymerase (2.5U/50 pi), TaqStart antibod) (0.55 Mg/5() ^1), dNTPs (0.2 niM each) and primers (0.2 mM each). The forward primer (5'-GCC'AGCT.CA TGT GCT ATA TGC TTG G-3') had a unique Sad site (bold), and the reverse primer (S'-GCA^AGCT^TC TGC ATA AAG GGC AC- 3') had a unique Hindlll site (bold), A four-step, hot start PCR reaction was performed. The conditions were as follows: 45 s at 95°C, followed by 10 cycles of 94°C, 45 s; 39,5°C, 60 s; 72°C, 60 s; 10 cycles of 94°C, 45 s; 38.5°C 60 s; 72°C 60 s; 10 cycles of 94°C, 45 s; 55°C, 60 s; 72°C, 60 s; 20 cycles of 94°C, 45 s; 54°C, 60 s; 72°C 60 s; and a final extension for 5 min at 72°C. Samples were rapidly cooled to 4°C. A single 142-bp PCR product was cloned into pCR^*^II, excised with appropriate restriction enzymes and directionally subcloned into the Sacl-Hindlll sites ofthe pSCREEN-lb(+) expression vector. Insert orientation was confirmed by sequencing in both directions by the dideoxy chain termination method.

Recombinant protein with a T7-tag, a His-tag and an S-tag at its NH2-terminus was induced by exposure to IPTG (1 mM) and purified by metal chelation chromatography. Briefly, recombinant His-Tag protein was eluted under native conditions (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), dialyzed against water to remove excess salt, and lyophilized. The protein concentration (1.05 mg/ml) was determined according to Dowry et al. (1951), using BSA as the standard. The leader sequence containing the T7-Tag and the His-Tag was removed by digestion with biotinylated thrombin. Biotinylated thrombin was separated from the remaining S-Tag protein with streptavidin-agarose.

2.2.3 Cloning ofthe RING Finger Binding Partner A /Igtl 1 cDNA expression library was prepared using mRNA isolated from HRE- H9 cells (Li et al, 1989) and screened according to the procedure of Maegregor et al. (1990) to identify protein binding partners. Nitrocellulose filter replicas were prepared

31 from A.gtl 1 recombinants (= 3 x 10"* plaque-forming units/plate) and processed with 6 M guanidine-IlCL denaturation-renaturation (Singh cl al, 1989; 1988) to ensure proper protein folding, fhe filters were then screened with the recombinant RING finger protein (1 |.ig/ml) with /nS04 (10 pM) to maintain functional conformation (Fan and Maniatis, 1990; Keller and Maniatis, 1991), and the 17 Tag antibody alkaline phosphatase conjugate (1:10,000). ,\ single, plaque-pure clone was re-screened with the thrombin- clea\ed recombinant protein (1 pg/ml) in the presence of ZnSOa (10 \xM), and the S- protein alkaline phosphatase conjugate (1:5,000). ,*\ positi\e plaque was heated at 100°C for 10 min, then amplified in a 50 pi PCR reaction mix containing LA PCR buffer (IX), TaKaRa ExTaq DNA polymerase (2.5U/50 pi), TaqStart anfibody (0.55 pg/50 pi), dNTPs (0.2 mM each) and Xgtl 1 primers (0.2 mM each). Forward (5'-GGT GGC GAC GAC TCC TGG AGC CCG-3') and reverse (5'- TTG AC A CCA GAC CAA CTG GTA ATG-3') ?igtl 1 primers flank the insert sequence in the vector. Hot start PCR reactions were performed with the following conditions: 120 s at 95°C, followed by 35 cycles of 94°C 45 s; 54°C, 60 s; 72°C, 180 s; and a final extension for 10 min at 72°C. Test and control reactions were rapidly cooled to 4°C. A single 1584-bp PCR product was cloned into pCR™II, and sequenced in both directions by the dideoxy chain termination method. Four different Marathon RACE (rapid amplification cDNA ends) reactions were performed to obtain overlapping cDNA clones as described in the Results and Fig. 2.1. Poly(A^) RNA (1 pg) from HRE-H9 cells was used in the preparation of adaptor-ligated cDNA libraries according to manufacturers' protocol. For each PCR reaction, 5 pi of diluted cDNA library was mixed with 45 pi of a PCR reaction mix containing LA PCR buffer (IX), TaKaRa ExTaq DNA polymerase (2.5U/50 pi), TaqStart antibody (0.55 pg/50 pi), dNTPs (0.2 mM each) and primers (0.2 mM each). In the first reaction (Fig. 2.1), RACE primer pair 1 consisted of a forward gene-specific primer (5'-GTG CGT GGA CTC CCT ATG CTG TTT CCC-3'), and a reverse adaptor primer (API; 5-CCA TCC TAA TAG GAC TCA CTA TAG GGC-3'). A three-step, hot start PCR reaction was performed with the following conditions: 60 s at 95°C, followed by 5 cycles of 94°C

32 30 s; 72 A\ 60 s; 72 'C, 180 s; 5 cycles of ^M"C, 30 s; 7r'C 60 s; 72°C 180 s; 25 cycles of ^)4 X\ 30 s; 70 'C, 60 s; 72 "C, 180 s; and a final extension for 10 min at 72°C. Samples were rapidly cooled to 4 'C A single 1151-bp PCR product (Fig, 2,1) was cloned into pCR^KMl-TOPO, and sequenced in both directions by the dideoxy chain termination method. In the second reaction (Fig, 2,1), RACE primer pair 2 consisted of a degenerate forward primer (5'-GAQ AAP ACN GGN ACN QTN ACN-3'; Q=pyrimidine, P=purine, and N = A. T, G or C). and a reverse gene-specific primer (5'-CGC TTT CTG CAG TGG TGC C\T ACG ACA GC-3 ). A four-step, hot start PCR reaction was performed with the following conditions: 30 s at 94°C, followed by 5 cycles of 94°C, 5 s; 65°C 240 s; 5 cycles of 94°C. 5 s; 60°C 240 s; 5 cycles of 94°C, 5 s; 55°C, 240 s; 20 cycles of 94°C, 5 s; 50°C, 240 s and a final extension for 10 min at 68°C. Samples were rapidly cooled to 4^0. A single 1185-bp PCR product was cloned into pCR®II-TOPO, and sequenced in both directions by the dideoxy chain termination method. The third reaction actually consisted of a primary reaction followed by a nested secondary reaction (Fig. 2,1), In the primary reaction, the RACE primer pair consisted of the forward API primer and a reverse, gene-specific primer (5'-CAG TGT GAC AGA GAC TGA CTG C-3')- The hot start PCR reaction was performed with the following conditions: 30 s at 94°C, followed by 30 cycles of 94°C, 5 s; 62,5°C, 240 s; and a final extension for 10 min at 68°C, Samples were rapidly cooled to 4°C, Products from this reaction were used in a nested PCR reaction with RACE primer pair 3 (Fig, 2,1). which consisted of forward adaptor primer 2 (AP2; 5'-ACT CAC TAT AGG GCT CGA GCG GC-3') and a reverse gene-specific primer (5-CCT TCT GAA GAA TCT GGT GTC GGT CC-3'). The hot start PCR reaction was performed with the following conditions: 30 s at 94°C, followed by 25 cycles of 94°C, 5 s; 66.5°C, 240 s; and a final extension for 10 min at 68°C. Samples were rapidly cooled to 4°C. A single 992-bp PCR product was cloned into pCR®II-TOPO, and sequenced in both directions by the dideoxy chain termination method.

33 The fourth reaction also consisted of a primary reaction followed by a nested secondar) reaction (Fig, 2.1), In the primary reaction, the RACE primer pair consisted of forward API, and a re\erse gene-specific primer (5'-CAA GGC CAC CAC TTC GAA C\\.\ C\ f .A.'V.V C.\Ci G-3 •). A six-step, hot start PCR reaction was performed with the following conditions: 30 s at ^)4 t\ followed by 5 cycles of 94°C, 5 s; 70°C, 150 s; 5 e)eles of ^)4X\ 5 s; 69^^G, 150 s; 5 cycles of 94°C, 5 s; 68°C, 150 s; 5 cycles of 94°C 5 s; 67°C 150s;5eyelesof94"C, 5s;66°C 150 s; 15 cycles of 94°C, 5 s; 65°C 150 s; and a final extension for 10 min at 68°C Products from this reaction were used in a nested PCR reaction with RACE primer pair 4 (Fig. 2.1), which consisted of forward AP2, and a rexerse gene-specific primer (5-GTC TCA ACC AAT CTT CGT ACC CCT G-3'). A six-step, hot start PCR reaction was performed with the following conditions: 30 s at 94°C, followed by 5 cycles of 94°C, 5 s; 68°C 120 s; 5 cycles of 94°C, 5 s; 67°C, 120 s; 5 cycles of 94°C, 5 s; 66°C, 120 s; 5 cycles of 94°C, 5 s; 65°C, 120 s; 5 cycles of 94°C, 5 s; 64°C, 120 s; 10 cycles of 94°C, 5 s; 63°C, 120 s; and a final extension for 10 min at 68°C. Samples were rapidly cooled to 4°C. A single 314-bp PCR product was cloned into pCR®II-TOPO, and sequenced in both directions by the dideoxy chain termination method.

2.2.4 Genomic Cloning An amplified rabbit genomic library in the EMBL3 SP6/T7 vector was screened with the 992-bp PCR product ofthe third PCR reaction described above and in Fig. 2.1. Nitrocellulose filter replicas were prepared from >.gtl 1 recombinants (=3x10 plaque- forming units/plate) and screened with the random-prime labeled cDNA (specific activity = 3-6 x 10^ CPM/pg). A genomic clone i~ 14-kb) was isolated and used as template in a PCR reacfion. A region ofthe genomic DNA (150 ng) was amplified in a 50 pi PCR reaction containing PCR buffer (1X), magnesium acetate (1.1 mM), Tth DNA polymerase (0.1 U), TthStart antibody (0.01 pg/pl), dNTPs (0.2 mM each) and primers (0.2 mM each). The forward (5'-CAG GGG TAC GAA GAT TGG TTG AGA C-3') and reverse (5'-CTG TCA GCG TAC CCG TTT TAT CTG TAA ACA C-3') primers matched sequence in the cDNA. The hot start PCR amplification reaction was performed 34 as follows: 30 s at 94'C, followed by 5 cycles of 94't', 5 s; 67°C, 360 s; 5 cycles of 94X, 5 s; 66''C, 360 s; 5 cycles of 94"C 5 s; 65°C, 360 s; 5 cycles of 94°C, 5 s; 64.5°C, 360 s; 20 cycles of ^)4 C, 5 s; 64A\ 360 s; and a final extension for 10 min at 68°C. Samples were rapidly cooled to 4°C A single 742-bp PCR product was cloned into pCRvK^ll-TOPO, and sequenced in both directions by the dideoxy chain termination method.

2.3 Results 2,3.1 Cloning ofthe RING Finger Binding Protein In order to test the hypothesis that the RING domain is involved in unique protein-protein interactions, the region ofthe RUSH cDNA that encodes the RING finger motif (amino acids 757-798) was isolated by the polymerase chain reaction (PCR) with primers that contained unique restriction enzyme sites, A single 142-bp PCR product was subcloned into pCR^'^II, excised with appropriate restriction enzymes and directionally subcloned into Sacl-Hindlll sites ofthe pSCREEN-lb(+) expression vector. Insert orientation was confirmed by sequencing in both directions by the dideoxy chain termination method. The resultant recombinant protein with a T7-tag, a His-tag and an S- tag at its NH2-terminus was used in a novel cloning strategy (Maegregor et al, 1990) to identify protein binding partners. Filters with recombinant proteins from a "kgt-X 1 expression library prepared from HRE-H9 cells were denatured-renatured with guanidine hydrochloride to insure proper protein folding (Singh et al, 1989, 1988). The filters were then screened with the recombinant RING finger protein in the presence of ZnS04 (10 pM) to maintain functional conformation (Fan and Maniatis, 1990; Keller and Maniatis, 1991). The single phage clone (1.6-kb cDNA insert), depicted in Fig. 2.1, was identified with the T7-tag antibody alkaline phosphatase conjugate. Because the His-tag is known to bind heavy metals, it was removed along with the T7-tag by enzymatic cleavage with thrombin. The tmncated recombinant protein with its S-tag was then used with the S- protein alkaline phosphatase conjugate to confirm the identity ofthe positive clone in a secondary screen.

35 fhe cDN,\ insert in the phage clone was amplified by PCR and subcloned into pCR^"^!!. Sequence anal) sis revealed the presence of a partial cDNA that contained a single open reading frame and a TAA stop codon, but lacked an initiator codon. Northern anal) sis of rabbit endometrial mRNA with this clone revealed a strong 5.3-kb band. As shown in Fig, 2,1, MarathonT^» RACE (rapid amplification of cDNA ends) was used to extend the 5-end ofthe cDNA and to obtain the entire 3'-end ofthe cDNA including the poK .\ tail. The 5'-region, which encodes the initiator methionine, is missing. The message at 5,3-kb is quite long and in the event of not obtaining full-length cDN.A in the re\ erse transcription step, any library subsequently made from these products will lack the 5' end ofthe message. This can technically be overcome by using the SM.ARTTMRACE cDNA synthesis technology to prepare the library. Briefly, this technolog) employs the terminal transferase activity ofthe enzyme MMLV RT that allows the addition of dC residues to the 3' end ofthe first-strand cDNA. The SMART oligo, that has a terminal stretch of dG residues, anneals to the dC-rich cDNA tail serving as an extended template for RT. Since this dC-tailing activity ofthe enzyme is most efficient when the enzyme has reached the end ofthe message, the SMART technology selects for full length RNA. 1 used the SMART RACE kit manufactured by Clontech to no avail. Fresh high qualify polyA"^ RNA was isolated from uterine endometrium and used to prepare new libraries but I still did not obtain the 5' end ofRFBP message. Therefore, repeated attempts to obtain this sequence using 5' Marathon'^'^ RACE and SMART''''^RACE cDNA synthesis systems met with no success.

2.3.2 ClustalW Alignment and Hydrophilicity Analysis ofRFBP Identify it as a Type IV P-fype ATPase A computer search of sequence databases (SWISS-PROT + PIR + PRE and GenBank) was conducted with the composite cDNA sequence (4286-bp) using the BLAST 2.0.12 program at the National Center for Biotechnology Information. This search revealed the predicted RFBP protein is 50% similar and 34% identical to Type IV P-type ATPases from Bos taurus (protein ID AAD03352), Mus musculus (protein ID AAB18627) and Saccharomyces cerevisiae (protein ID L01795). This alignment showed

36 the conserved RFBP and the partial cDNA sequence for human KIAA0956 (GenBank accession number AB023173) are also 92% identical using the BLAST algorithm for pair-w ise DNA-DNA sequence alignment, while the amino acid sequences are 93% identical and 95% similar. Such comparisons indicate that KIAA0956 is the human ortholog ofRFBP. A position-specific iterated BLAST (PSl-BLAST) program (Altschul cl al, 1997) which is sensiti\e to weak but biologically relevant sequence similarities identified 597 hits. .All ofthe hits were ATPases, and 289 of them had statistically significant expectation (e) values greater than the 0.001 threshold. Additionally, once the BL.AST searches showed that RFBP is similar to Type IV P-type ATPases, ClustalW alignment (Thompson et al, 1994) was used to align RFBP sequences with the published sequences for Bos taurus. Mus musculus and Saccharomyces cerevisiae (Fig. 2.2). This not only helped in the identification of conserved regions among the proteins but when used in conjunction with data from hydrophilicity analysis (Fig. 2.3, discussed later), putative transmembrane segments were identified in RFBP. The ClustalW alignment indicates that RFBP has all ofthe diagnostic structural features typical of P-type ATPases. These are the consensus sequences for seven of eight core segments that include the highly conserved phosphorylation site [DKTGTLT], characteristic of P-type ATPases (Moller et al, 1996) and the TGDN mofif in region F that is the putative ATP- binding domain (Lutsenko and Kaplan, 1995); along with nine transmembrane domains (TD). It is important to note that region D which contains transmembrane domain 4 is absent from the RFBP cDNA sequence. Transmembrane domain assignments (Fig. 2.3) were confirmed by analyses with the SOAP algorithm derived by Kyte-Doolittle (Kyte and Doolittle, 1982), the Argos et al (1982), transmembrane analysis, the Tmpred program (www.ch.embnet.org) and the SOSUI system (http://azusa.proteome.bio.tuat.ac.jp/sosui/). However, to avoid confusion, the domains were numbered consecutively in Fig. 2.3. The composite cDNA sequence (4286-bp) and the predicted amino acid sequence (1107 amino acids) for RFBP (Genbank accession number AF236061) with the core regions and transmembrane domains are shown in Fig. 2.4. The incomplete protein has a predicted molecular mass of 126 kDa.

37 2.3,3 RFBP gene lacks Region D .Ml known P-type ATPases contain an even number of transmembrane passes with four of them highh conserved at the NIL-terminus (Axelsen and Palmgren, 1998; Geering, 2000; Lingrel and Kiint/weiler, 1904), I bus, it was important to determine whether or not region D, which is an evolutionarily conserved domain and is missing from the cDN.A, was also missing from the gene. Primers that recognize cDNA sequence on either side of Region D, in transmembrane domain 2 and Core Region E, were used in a PCR reaction with genomic DNA. Analysis of PCR reaction products by agarose gel electrophoresis is shown in Fig. 2.5A. Gel electrophoresis with MetaPhor agarose and ethidium bromide fluorescence allowed the visualization of a single 742-bp reaction product. Genomic sequence data provided in Fig. 2.5B has 100% identity with the corresponding region ofthe cDNA. These data show that region D which contains transmembrane domain 4 is absent from the RFBP gene, and confirm that RFBP is not a splice variant.

2.4 Discussion P-type ATPases comprise an evolutionarily diverse superfamily of nearly 200 ATP-hydrolysis-driven ion pumps (Axelsen and Palmgren, 1998; Scarborough, 1999). Structiually related to hydrolases (Aravind et al, 1998), P-type ATPases are thought to share a common catalytic mechanism with these enzymes. Because these integral membrane proteins show very little similarity to each other (<15%), they were originally described as PI ATPases (heavy metal pumps), P2 ATPases (nonheavy metal pumps and phospholipid translocases) and P3 ATPases (bacterial K'^-pumps). However, when arranged phylogenetically according to substrate specificity and sequence alignment, P- type ATPases can be divided into five main groups (Fig. 2.6). The groups are designated Type I (heavy metal pumps). Type II (Ca^^, Na^K^ and HVK^ pumps). Type III (H^ and Mg^"" pumps), Type IV (phospholipid pumps) and Type V (no assigned substrate specificity). Type IV, the most recently discovered group, is found only in eukaryotes, and appears to be very distinct from the metal ion transporters that dominate this enzyme class. The absence of Type IV from prokaryotes supports the hypothesis that P-type

38 ATPases are e\ ol\ ing at a variable rate, and that Type IV enzymes have evolved more recently than their counterparts (Axelsen and Palmgren, 1998). .Ml working models of membrane topology for P-type ATPases, identified to date, contain eight or ten clearly defined transmembrane passes with four highly conserved spans at the NHi-terminus. Collectively, these transmembrane helices anchor the protein in the lipid biUner, orient the hydrophilic domains ofthe protein at the membrane surface, and assemble into channel-like structures to regulate transport (Fig. 2.7). The hydrophilic portions ofthe protein which include its NH2- and COOH-termini and two loop segments are located on the same side ofthe plasma membrane and therefore in the same sub-cellular compartment (Moller et al, 1996). As depicted in Fig. 2.7, this topology preferentially exposes the small, strongly hydrophilic loop (Regions A-C) between transmembrane domains 2-3, and the large conformationally flexible loop (Regions D-H) between transmembrane domains 4-5, to the cytoplasm. Regions A-C have been assigned a role in energy transduction that may be as simple as maintaining the stability ofthe ATPase stmcture. Transmembrane domain 4 (TD4) is postulated to be directly involved in the energy transduction mechanism. The sequences that link TD4 to the phosphorylation site, i.e., regions D and E, are two ofthe three most highly conserved regions in ATPases. Mutafions in this region cause a significant reduction in the transport capabilities of H""-ATPase (Ambesi et al, 1996). The conformationally flexible loop that accounts for 45% ofthe polypeptide contains highly conserved phosphorylation and ATP-binding sites. The function ofthe phosphorylation site in all P-type ATPases is the same, i.e. the transfer of y-phosphate from ATP to the aspartate residue in the phosphorylation site. In fact, P-type ATPases were named for the participation of a high- energy aspartyl-phosphoryl-enzyme intermediate in their catalytic cycle (Pedersen and Carafoli. 1987). Phosphorylation ofthe aspartate (D) residue in the invariant sequence DKTGT(L/I)T distinguishes P-type ATPases from V-type and FoFi-ATPases. All known type IV P-type ATPases also have the same general characteristics of P-type ATPases. However, the newly identified RFBP differs from all previously described Type IV P-type ATPases. The most striking feature of this putative phospholipid pump is an odd number of transmembrane domains resulting from the absence of Region D and TD4.

39 Because ofthe highh conserved nature ofthe NFL-tcrminus, the absence ofthe fourth membrane-embedded domain alters both the orientation ofthe adjacent hydrophilic domains and the determinants of transport specificity. Deletion of this region alters the conformational dynamic that functionally links the transport site in the membrane with the site of .\TP h)drolysis, and strongly suggests that RFBP is not involved in phopholipid transport. In the larger scheme of evolutionary advantage, altered substrate specifieit) requires a change in structural constraint resulting from a dramatic change in primar) structure (,\xelsen and Palmgren, 1998). Such a change in substrate specificity is likel) to result from the absence of Region D and the PEGL motif, which is considered to play a critical role in energy transduction (Clarke et al, 1989). M) first thought was that the endometrial RFBP might be the product of an altemati\e splice e\ent. Once considered to be the exception to the mle (one gene, one protein), altemative splicing is known to regulate approximately 5% of all genes (Sharp, 1994). It is an economical way to produce different products from the same gene (McKeown, 1992; Smith et al, 1989). A survey on intron and exon length by Hawkins (1988) indicated that although introns in vertebrates vary in size from 40 to greater than 3000 nucleotides, the largest single size range is 80-90 nucleotides. Region D and its included transmembrane domain 4 are encoded by 72 nucleotides that could easily participate in the simplest of altemative splicing events, i.e. splice/don't splice. However, sequence from genomic clones confirmed that Region D and TD4 are absent from the RFBP gene. These results support a working model for membrane topology in which the NH2- and COOH-termini ofRFBP are located on opposite sides ofthe membrane. The impact of this altered structure, on protein funcfion, calls for closer scrutiny ofthe role ofthe conserved motifs in mediating RFBP function.

40 Primer Pairs >i RT-PCR > < RACE 4 RACE 3 > < RACE 2 • •^ RACE1 TAA 1 ® Original clone ^23 ••1 Adaptor II Adaptor 1

Adaptor • IV RACE Products Orientation Overlap (bp) Size (bp) 1 3' 283 1151 II 5' 360 1185 III 5' 125 992 IV 5' 172 314

Figure 2.1. Strategy for RACE PCR Reactions. The open box depicts the original 1.6-kb clone with the stop (TAA) codon. Large arrowheads mark the locations ofthe primer pairs used in the RACE procedure. The sizes ofthe overlaps between known sequence and additional sequence obtained as single reaction products by 5'- and 3'-RACE are provided in base pairs (bp). Region C and the putative phosphorylation (P) site are provided for orientation. The poly A tail (A23) is indicated at the end ofthe 3'-RACE product.

41 TDl iW Jt'.'-^p _GjiqHjr7 E Q F B B AM Y P 1 IIM1VQ|L M I •Tits TPIPRFIY Q F B B A H P F I I A L L Q Q I P 'Its /•^>tori:.'i^SLV,'feVA7?>41r\* iY.'-f\-7Sf^ A TPLPK FLF E T[H]K AH PPL T|g[All Q Q V P T F L P F L Q F R E A H F F L I L q Q p

iW TD2 •Vc? PIT - FPT|S P P LJF IP V I T VT A I K Q G Y EPJV DViPTGBTIIL VPllP I lA VA A I K E I E D I D^V •PTGBTTTLVPLLF I LAVAAIKE I E D I Ss£t-/fsr.i?ff?^y?sce;-s\-7S?d^ lilv • P T N B T T T IG T|L Lfvlv LfTlvs AME E I E D I D V S P T R Y T T PLLF L V AIKE I E D I

.'W .;W 300 LIB H N SDH E V H G A P V Y V V B G G L V K T|R" S K H I 3?s r.si.'ri/s FB H K A D H A VH K K Q T 0 V L B H G A V E I VH - X B H K A D H A VH K K Q T 0 V L B N G A V E I VH X B[A N S DfK E L H N S T A E I|F 8 E A H D D F V|E K R - = K R H D N V N V R G V

REGION A ,:iV JSO fffffP R G M H L VG D I V I|A K D F|P AD|L|V1|I I 1|D R L Sas Tsijnjs VG D I V I K K IPADTVLLISSEP Hus Musculijs VG D I V I K G K IPADTVLLISaEP Sacctt$romyc&s cerev?s?s$ VG D I I V K 8 E IFADT I I LfSBEP V V G D I V I K IPADTVLLSSSEP REGION B JW RFBP D G sfc]H|V|T|T A|g|lDGETHLX|T [HIV A V P I A L Q Bos Taurus MCTIETS HLDGETHLEIRQ G I S I K Nus Nusculus- MCTIETSHLDGETHLKIRQ G T S I K Sscchsromi/ces cerevrsfst LCYIETAHlDGETMtE IKQ g R V T A F I CYIET NLDGETNLKI P T REGION C «y •iio «V PFBP T Q M E E I V RjP L G P LLLBGARLKHTK Bos Taurus D G g T V P I 6 A DQ I LLBGAQ LBHTOV Nus Nfjsculus D G G T V P L G A DQ I LLBGAQ LBHTQV Saccharomyces corovisiae Q I P Lfg P P <^M I L B G A[T1L B HT[A1V P L G DQ LLRGA LRNT V

*y 4^ 450 PFBP I F GVAVTTGM|E|TE M|A L |H|Y K |l Kg Q[X A VIE K Bos Taurus V H G I VVTTGHD TXLMQHS Tf P IP L X N VE R Nus Musculus V H G I VVTTGHD TKLMQHB Tf P L E N VE R Saccharomi/ces caravisiae I F GLVI FTGHE TKLLRHATA P I X A VE K VVYTGH TKL T S K V E

Figure 2 2 ClustalW Alignment ofRFBP with Type IV P-type ATPases Sequence of Type IV P-type ATPases from Bos taurus (protein ID AAD03352), Mus musculus (protein ID AAB 18627) and Saccharomyces cerevisiae (protein ID L01795) were obtained from the database. Regions of alignment containing core regions A-H (red) and transmembrane domains (TD) 1-10 (blue) are depicted here. Note absence of region D (pink) containing TD4 (purple) that is present in other Type IV P-type ATPases

4^ •fey TD3 •«y PFBP 8 M T FlLJTmY L HI L I S|E|A I I ST IIL KIY T |V^ Bos Taurus QILILPCILlAMSLVCBV^ S A A/us A/uscu/us <)IL ILPCILIAMSLVCBVG S A S$cc.^ro.'y7i,tc^s c^ra w.'s.'ae Qi I[R1LPT VLI[V1L(T|LI S«IG H V Vl ILF ILI L SG

•rtV 500 TD4 P.'^BP E K sro Bos .^aurus H n G R D V 7L H L IT Y G G A IT H P G L Hip LIP I ILF A/us A/usi^u.'its H s G K V V TL H L H Y G G A g H P G L H F L T F I ILF S^cc/)aromi,tiias cerawfsraa D A K H L 8 T L Y L E G T N K A G L P F K D.P LIP V ILF, Y L L A F F L T F ILF

REGION D 5lV SM PFBP |VD|E_ BOs Taurus H HL I P I 1 L L V TJL E V V xir Q A Y I H VP L P M .'^/us A/uscu/us N HL I P I • L L VTL EVVXF Q A Y I N VD L P M Ssn'c.'Siaromycas c^raK's?aa g HL V P I • L F VT V E L I X Y Q AF I G[g]p L P L N L P I g L V T E K Q A I V D L REGION E Phosphorylat ion 550 5!k. PFBP B H • g[K" T V F T P X T Bos Taurus A M B T 1 N V E L G Q V TIPS D K T A/us A/uscu/us A M B T fl H E E L G Q V T I P • P X T Saccharomi/ces caravisfae T V B T • V E E L G 0 I TIPS PET R T g ELGQV YIFgDKT domain sso 590 i?00 PFBP G T L T E HJE |HQ F R E C g l[HjGi;i|KjT]Q N G R L V P E Bos Taurus G T L T H V HQ F K K CTlAGVATG g 0 F A/us A/uscu/us G T L T c HV MQ F X K CTIAGVATG S Q F Saccttaromyc-es c-arevisiaa 6 T L T R H I ME F K g c s I AG[H1C T|I D K I P E GTLT N MQFK I A G

L I L Bos Taurus A V T HHJ R Ag TS VQHB L L X L E E S L I E X H L L A/us A/uscu/ui- A V T H RAg Tg VQHB L L K L E E 8 L I E K H L Q L Saccharomtjcas caravisraa 8 I T N E|A A T T LfFlHB A E X L D E A1X11 L I E X HL I L 0 N R K L L I E K H L .- ) ATP binding REGION F S50 960 domain 0^0 PFBP LGATAVEPRLQPKVRIET lEAL R M A G I X V vv Bos Taurus LGATAIEPXLQPQVPET lETL M K A I X I V I A/us A/uscu/us LGATAIEDKLQPQVPET lETL M K A D I X I V I Sacc/iaromi/ces carsvisfaa IGATAIEPXLQ P[G1V P E T \^1 L Q E A G I X I vv I K I V

080 090 900 PFBP L T G P X H E T A V|g|V g L • C G H F H R T MN 1 L[EjL T N Bos Taurus LTGPXQETAIHI • C K L RRKNMGMIVIH A/us A/uscu/us LTGPKQETAIHIG • C R L L [K_RN M G M I V I H Saccharomycas cerav/s/aa L T G P R Q E r~lA I H I G • C R L L gED|MNLLIIH LTGDKQETAINIG gC M I N Figure 2.2 (continued)

43 REGION G 9.^? .00 990 AVlLCCBMAPLQXA K V I R L I X I g P E X P I T I G B&f Tauri.t.f AVICCBVfPLQXg E V V E M V X K Q - V X V I T L A .'^/us .'^/uscu/us AVICCBVfPLQXS E V V E MVX K Q - V X V I T L A Ssii^.^ro,'??iti:asca.'-aK's.'aa AVICCBVIPLQXA L V V K MVX R K - 8 8 8 LfTlL A AVICCRVgPLvK V V M V K K I T L A

moo w/o }020 c V PGAHPVMIQ|E|AHV6 I G I|M[G E|R R|Q AATR' JSV Taurus PGAHPVtMIQT AHVGVG If E G L Q A A A/us .Muse u/us PGAHPV0MIQT AHVGVG IIG E G L QA A Sacctsarcii^icas cars K's.'aa T|q AHPVMIQAAHVGVG IJG E GMQ A A D G A H r VSMIQ AHVGVG18G E G Q A A REGION H !000 )040 !050 P.^ffp HBPTAIARFXFL8K L L|F V H GjH F Y T)I|BII A T L Bos Taurus ••PT8lAQPXYLX H LLMVHGAV IT T N B[Gji K C A/us A/usi'u/us ••PT8 lAQFEYLE H LLMVHGAV H T N H V • K c Sscc/naromi/cas carai'?s?aa • A PRIA Lf^lQ F E F L XK LLL VHGSV 8 TQ B I • V A 8 g D Y LK LL VHG V Y TD5 rooo WFO 1000 V|Q|T|F PTXHVCFITP F L Y Q Y C L PTIOJQ T L Y Bos Taurus I L T C T X H I T I fVF F • G Q I L F A/us A/uscu/us I L T C TX H I T I F P t G Q I L F Sacc/iaromt/ces cerav?s/ae I L T g T K Hfr" TM Y F • G Q 8 _IJM I L Y F Y K N V F 8 G Q TD6 f090 f/00 /f!0 PFBP g V Y L T L TH I c F T g L P^L I Y g L L E Q H I D P H Bos Taurus R VC I G L TH V M F I AM P P L T L 6 I PER g c R K E A/us A/uscu/us R VC I G L TH V M F T A M P P L T L G I PER 8 c R K E Saccharofngm- carsvisiaa 8 VT M 8 F|TH L F P T|V V|P PfF V I G V P D Q F V g g R V L Y H FT PPL G F E TD7 Hi^yj tlOO }140 PFBP I L H J

f/50 f/60 IfFO PFBP [F g|R • F IlF L F G g Y F IJG K D|A g L L G GQ M F G H| Bos Taurus L P Bf V I L P V F P L K L "5 TG T V F E N G R T g P Y L PHI V ILF V F P L K L Q TG T V F G N G K T S P Y

A/us A/uscu/us i j GIE A P H Ssco/faromi/cas oaravtstaa F PHI A I V F I G T I L YIR TGfF A L N M H F H 8 Y G TD8 1100 1190 f^VO PFBP V J T G T L V F T V M V I T VT V T\M_ A LIE T F VT __I N Bos Taurus L L I 9 V r r L E T Y VT vT g A/us A'/uscu/us LLL G N F VTT F VVITVC LXA G LET Y VT VF S Saccharomt/cas carav/s/aa V g V Gfv T VTT T g]V_l]T|v|L GIX A A jJTlT HT1VT[F|F T G . V Y T V I T V K A LET V T V F Figure 2 2 (continued)

44 TD9 l.i'10 /.^'.kV /.^'•SO PFBP BL|V T|VG • I I F Y FIVF18 L FpTlG G|I L V P F L G|g Cj IT Bos Taurus H I A I VG • I A L VV V P P '3 I T g • L P A V P M A P[D A/us .Muscu/us H I A I VG • I A L V V V F F '•J I T g • L W P A V P M A P D SsiTharomifiras cera\'7SFaa LJI A IF|G t L L F VL I P P P I T A • I F P H A IT I g R H I A I V G 8 I V V F F I Y TD 10 IJ-^? /.'i5y /J'^O Y F V F I L|V|1 f G|S A|V|F A I I M V VT C L F L P V Bos Taurus 8 O E A A MIL F • • 6 V F VM L L I PVA8LL LPV A/cts .Musi'u'i.{s _8 G E A A M[I F fl ff G V F V V L L IPVASLLLPV Sacc/isrc^yiA^scaraK'siaa Y Y[GJV V K H TlY_[G"[fl_G_V_F_V_L L I L P 1 [FJA L V[F|P[F^ M G L.ggGVFV P V L L D V Figure 2 2 (continued)

45 Hydrophilicity Plot +4- hydrophilic

+2 —

+1-

-2-E

-3 — hydrophobic -4' I i I I I I I I i i 100 200 300 400 500 600 700 800 900 1000 aa amino acid positiion

Figure 2.3. Hydrophilicity Profile. Hopp-Woods hydrophilicity analysis ofthe partial sequence of the putative P-type ATPase was obtained with the DNA Inspector program, version 3.16 using a window of 12 residues proceeding from the NH^-terminus to the COOH-terminus. A total of nine transmembrane domains (numbered brackets) were identified by their extreme hydrophobicity.

46 1 GCTGGGTTTTGACCCACCACATCAAAGTGACACAAGAACTATTTACATAGCCAACAGATT

1 LGFDPPHQSDTRTIYIANRF

61 TCCTCAGAATGGCCTTTATACACCTCAGAAATTTATAGATAATCGGATCATTTCATCTAA 21 PQNGLYTPQKFIDNRIISSK

121 GTATACTGTGTGGAATTTTGTTCCAAAAAATTTATTTGAACAATTCAGAAGAGTGGCAAA 41 YTVWNFVPKNLFEQFRR V A N

181 CTTTTATTTTCTTATTATATTTTTGGTTCAGCTTATGATTGATACACCTACCAGTCCAAT 61 FYFLI IFLVOLMIDTPTSPJ I Transmembrane Domain 1 2 41 TACCAGTGGACTTCCATTATTCTTTGTGATAACAGTAACTGCTATAAAACAGGGGTACGA 81 T S G I L P L F F V I T V T A I K Q G Y E~ Transmembrane Domain 2 301 AGATTGGTTGAGACATAACTCAGATAATGAAGTAAATGGAGCTCCTGTTTATGTTGTTCG 101 D W L R I HNSDNEVNGAPVYVVR

3 61 AAGTGGTGGCCTTGTAAAAACTAGATCGAAAAATATTCGGGGTATGCATCTGGTAGGTGA 121 S G G L V K T RSKNIRGMHLVGD Core 421 TATTGTTCGAATAGCCAAAGATGAAATTTTCCCTGCAGACTTGGTGCTTCTGTCCTCAGA 481 IVRIAKDEIFPADLVL L S S D Region A 481 TAGACTTGATGGTTCATGTCACGTTACAACTGCTAGTTTGGATGGAGAAACTAACCTGAA 161 R L D G S C H VTTASLDGETNLK Core Region B 541 GACTCATGTGGCGGTTCCAGAAACAGCAGTGTTACAAACAGTTGCCAATTTGGACACCCT 181 T H V A V P E TAVLQTVANLDTL

601 AGTGGCTGTGATAGAATGCCAGCAACCAGAAGCAGACCTGTACAGATTCATGGGACGAAT 201 VAVIECQQPEADLYRFMGRM

661 GGTAATAACTCAACAAATGGAAGAAATTGTAAGACCTCTGGGGCCAGAGAGTCTCTTGCT 221 VITQQMEEIVRPLG P E S L L L

Figure 2.4. Alignment of Nucleotide and Predicted Amino Acid Sequences for RFBP. In the predicted amino acid sequence, nine transmembrane domains are boxed, and Core Regions A-H are underlined. The putative phosphorylation site (DKTGTLT) in Core Region E, and the putative ATP binding domain (TGDK) in Core Region F are shaded. The antigenic site (TNQKSDSECAEQLRQL) used for antipeptide antibody preparation (shaded) is located in the conformationally flexible loop between regions F and G. Note Core Region D is missing. In the nucleotide sequence, the polyadenylation signal (AATAAA) is underlined.

47 7 21 TCGTGGAGCTAGATTAAAAAACACAAAAGAAATTTTTGGCGTTGCTGTATACACTGGAAT •41 RGARLKNTKEIFGVAVYTGM Core Region C 7 81 GGAAACTAAAATGGCATTAAATTACAAAAGCAAATCACAGAAACGCTCTGCAGTAGAAAA :61 ETKMALNYKSKSQKRSAV fe K

841 GTCAATGAATACATTTTTGACCATTTATCTTATAATCCTTATTTCTGAAGCCATCATCAG 181 SMNTFLTIYLIILISEAIIS Transmembrane Domain 3 901 TACTATCTTGAAATATACATGGCAAGCTGAAGAAAAATGGGATGAGCCTTGGTATAACCA 301 T I L K Y TWQAEEKWDEPWYNQ

9 61 AAAAACAGAACATCAAAGAAATAGTAGTAAGGTGGAATATGTGTTTACAGATAAAACGGG 321 K T E H QRNSSKVEYVFTDKTG Core Region E with Phosphorylation 1021 TACGCTGACAGAAAATGAGATGCAGTTTCGAGAATGTTCAATTCATGGCATGAAATACCA 341 TLTENEMQFRE CSIHGMKYQ site 10 81 AGAAATCAATGGTAGACTTGTACCCGAAGGACCGACACCAGATTCTTCAGAAGGAAATTT 361 E INGRLVPEGPTPDSSEGNL

1141 GTCTTATCTGAGTAGTTTATCCCATGTTAACAGTTTATCCCATCTTACAAGCAGTTCCTC 381 SYLSSLSHVNSLSHLTSSSS

12 01 TTTCAGAACCAGTCCTGAAAATGATACTGAACTAATTAAAGAACACGATCTCTTCTTTAA 401 FRTSPENDTELIKEHDLFFK

12 61 AGCAGTCAGTCTCTGTCACACTGTACAGATTAGCAGTGTTCAAACTGATGGCATTGGTGA 421 AVSLCHTVQISSVQTDGIGD

13 21 TGGTCCCTGGCAGTCCAGCCTGGCACCGTCGCAGTTGGAGTACTATGCGTCTTCACCAGA 441 GPWQSSLAPSQLEYYASSPD

13 81 CGAAAAGGCCCTGGTGGAAGCTGCCGCACGAATTGGCATTGTTTTTGTGGGCAATACTGA 461 EKALVEAAARIGIVFVGNTE

1441 AGAAACTATGGAAGTTAAAATACTTGGAAAACTGGAAAGGTACAAACTGCTTCATGTTCT 481 ETMEVKILGKLERYKLLHVL

1501 AGAATTTGATTCAGATCGTAGGAGAATGAGTGTAATTGTTCAAGCACCATCAGGTGAGAG 501 EFDSDRRRMSVIVQAPSGER

15 61 ATTTCTGTTTGCTAAAGGAGCCGAGTCATCTATTCTCCCCAAGTGTATAGGTGGAGAAAT 521 FLFAKGAESSILPKCIGGEI

Figure 2.4 (continued)

48 1621 AGAAAAAACAAGAATTCATGTAGATGAATTTGCTTTGAAAGGGCTAAGAACTCTGTGTGT 541 EKTRIHVDEFALKGLRTLCV

16 81 AGCATATAGACAGTTTACATCAAAAGAATATGAAGTGATAGATAGACGCCTGTTTGAAGC 561 AYRQFTSKEYEVIDRRLFEA

17 41 CAGGACTGCCCTGCAGCAGCGGGAAGAGAAATTGGCAGATGTCTTCCATTATATAGAGAA 581 RTALQQREEKLADVFH Y I E K

1801 .AGACCTGATATTACTTGGAGCTACAGCAGTGGAAGACAGACTGCAAGATAAAGTTCGAGA 601 DLILLGATAVEDRLQDKVRE

18 61 .AACTATTGAAGCATTGAGAATGGCTGGCATCAAAGTATGGGTACTTACTGGAGATAAACA 621 TIEALRMAGIKVWVLTGDKH Core Region F with putative ATP binding site 1921 TGAAACAGCTGTTAGTGTGAGTTTATCATGTGGACATTTTCATAGAACCATGAACATCCT 641 ETAVSVSLSCGHF H R T M N I L

19 81 TGAACTTACAAACCAGAAATCAGACAGCGAATGTGCTGAACAATTAAGGCAGCTTGCCAG 661 ELTNQKSDSECAEQLRQLAR Site for Antipeptide Antibody 2 041 AAGAATTACAGAAGATCATGTGATTCAGCATGGGCTGGTGGTGGATGGAACCAGCCTCTC 681 RITEDHVIQHGLVVDGTSLS

2101 TCTTGCACTCAGGGAACATGAAAAACTATTTATGGAAGTTTGCAGGAATTGTTCAGCTGT 701 LALREHEKLFMEVCRNCSAV

2161 GTTATGCTGTCGTATGGCACCACTGCAGAAAGCGAAAGTAATCAGATTGATAAAAATCTC 721 LCCRMAPLQKAKVIRLIKIS Core region G 2 2 21 CCCTGAGAAACCTATAACCATTGGCTGTTGGGATGGTGCTAATGATGTAAGCATGATACA 741 PEKPITIGCWDGANDVSMIQ

2 2 81 GGAAGCACATGTTGGAATAGGAATCATGGGTAAAGAACGAAGACAGGCTGCAAGAAATAG 761 EAHVGIGI M G KERRQAARNS

2 3 41 TGACTATGCAATAGCTAGATTTAAATTCCTCTCCAAATTACTTTTTGTTCATGGTCATTT 7 81 DYAIARFKFLSKLLFVHGHF Core Region H 2401 TTATTATATTAGAATAGCTACCCTTGTACAGTATTTTTTTTATAAGAATGTGTGTTTTAT 801 Y Y I R I A T L V Q Y FFYKNVCFI Transmembrane Domain 4 24 61 CACACCCCAGTTTTTATATCAGTTCTACTGTTTGTTTTCCCAACAAACACTGTATGACAG 821 ~T P Q F L Y Q F Y C LT] FSQQTLYDS

Figure 2.4 (continued)

49 2 521 TGTGTACCTGACTTTATACAACATTTGTTTTACTTCCCTACCTATTCTGATATATAGTCT 841 IvYLTLYNICFTSLPILIYSL Transmembrane Domain 5 2 581 TTTGGAACAGCATATAGACCCTCATATATTACAGAACAAGCCTACCCTCTATCGAGACAT 861 L E QHIDPHILQNKPTLYRDI

2641 TAGTAAAAATCGTCTGTTAAGCATTAAAACATTTCTTTATTGGACCATCCTGGGTTTCAG il SKNRLLSI K T F LYWTILGFS Transmembrane Domain 6 27 01 TCGATCCTTTATTTTCCTTTTTGGATCCTATTTTCTAATTGGC/^AAGATGCATCTCTGCT 901 R S F I F L F G S Y FLIGKDASLL

27 61 TGGAAATGGCCAGATGTTTGGAAACTGGACATTTGGCACTTTGGTCTTCACAGTCATGGT 921 G N G Q M F G N W TFGTLVFTVMV Transmembrane Domain 7 2821 TATTACAGTCACAGTAAAGATGGCTCTGGAAACTCATTTTTGGACGTGGATCAATCATCT 941 ITVTVKMA L| E THFWTWIN ^ L

2 881 TGTTACCTGGGGGTCTATTATATTTTATTTTGTGTTTTCTTTGTTTTATGGAGGGATTCT 961 VTWGS I IFYFVFSLFYGGIL Transmembrane Domain 8 2941 CTGGCCCTTTTTGGGTTCCCAGAATATGTACTTTGTATTTATTCAGCTGGTATCAAGTGG 981 W P F L GSQNMYFVFI 3 L V S S G

3 001 TTCTGCTTGGTTTGCCATAATTCTTATGGTTGTTACATGTTTGTTTCTTGATGTCATGAA 1001 SAWFAI ILMVVTCLFLDV|M K Transmembrane Domain 9 3 0 61 GAAGGTATTTGACCGACAACTCCATCCTACAAGTACTGAAAAGGCACAGCTTACTGAAAC 1021 KVFDRQLHPTSTEKAQLTET

3121 AAATTCAAGTATCAAGTGCGTGGACTCCCTATGCTGTTTCCCAGAAGGAGAAACAACGTG 1041 NSS IKCVDSLCCFPEGETTC

3181 CACATCTGTTAGAAGAATGCTGGAACGAGTTATAGGAAGATGTAGTCCTACCCACATCAG 1061 TSVRRMLERVIGRCSPTHIS

3241 CAGATCATGGAGTGCGTCGGATCCTTTCTATACCAACGACAGGAGCATCTTGACTCTCTC 1081 RSWSASDPFYTNDRSILTLS

33 01 CACAATGGACTCGTCTACTTGCTAAGGGGGCAGTAGTACTTTGTGGGAGCCATTTCACCT 1101 T M D S S T C •

33 61 CCTGTCCTAAAATTGAGTACGATCATCCCGTGAATGGCCACATTAGCTCTGAAATCCCTT

3421 TCTGAAGCCTCGAGTAGTTCACCCCCATACAGAGTTACAGTGGCAAACAATCAGAAAGCC Figure 2.4 (continued)

50 3481 TAACATGAATCCTGCACATTCCACCTTGAGCCTGAACATGCCACAGTTTGTGAATAAAGC

3 541 AGACATTTTCTATCTCTTTGTCTGGATTTGTCCCTTGTGCTTATGGGACTCCTCATGGCA

3 601 TTTCAGCCTGTTGCTGAGGCCACTATATTTTAATATCAAGAGAAAAAGAGCGAGAATTCC

3 6 61 TCCTTCATGTGTATTTTTTAGTATTAGCTTGGTTATTGACTCTATTTAAATCTGCTTCTG

37 21 TAAATTATGCTGAAAGTTTGTGTTGAGAACTCTATTTTTTTATTAGAGTTATATTTGAAG

37 81 CTTTTCATGGGAGAAGCTAACATGAATAGTCAGGAATCTCGGTCCCTGCGTGACCCTTGG

3 841 TAATTCAGTAGTGCTTTAGAAGTTTGCAGGTGAATTTAGGAGAATGCATTTCCAACCATT

3901 ATTTACTGCAGTCTGGGTAGTGAATTGATACTGGTAACAGATTTCTCCTAAAGTACTGTA

3 961 ACACAACCAGTAAAGTTAGCACCATATAAATGCAAAGGGTGTAGAATATCTTACATATAA

4021 ATCAGGAATCAGGTCCATTCACC AAATTTCAAACTGATGTTTACTATTTTTGTCACAGAA

4081 TATGAAGAACCCATAGTGGGTAAATTGGGTACCATTAGTATATTATTAATTTAATGTGTT

4141 ATCATTGAATCTTTTGCATGCTTTAAGTATCTAAATTTGCCTTATTTTCTCTGAAGCTTC

4201 TGTTCATAGTATTTTATGATAATGTAAAGTTCAACCATATCAATAAAAACAATTTTGGAC

42 61 AGTAAAAAAAAAAAAAAAAAAAAAAA

Figure 2.4 (continued)

51 B Size 1 2 5'-CAGGGGTACGAAGATTGGTTGAGACATAACTCAGAT AATGAAGTAAATGGAGCTCCTGTTTATGTTGTTCGAAGT 3000 GGTGGCCTTGTAAAAACTAGATCGAAAAATATTCGGGGT 2500 ATGCATCTGGTAGGTGATATTGTTCGAATAGCCAAAGAT 2000 GAAATTTTCCCTGCAGACTTGGTGCTTCTGTCCTCAGAT AGACTTGATGGTTCATGTCACGTTACAACTGCTAGTTTG GATGGAGAAACTAACCTGAAGACTCATGTGGCGGTTCCA 1500 GAAACAGCAGTGTTACAAACAGTTGCCAATTTGGACACC CTAGTGGCTGTGATAGAATGCCAGCAACCAGAAGCAGAC CTGTACAGATTCATGGGACGAATGGTAATAACTCAACAA 1000 ATGGAAGAAATTGTAAGACCTCTGGGGCCAGAGAGTCTC TTGCTTCGTGGAGCTAGATTAAAAAACACAAAAGAAATT 750 TTTGGCGTTGCTGTATACACTGGAATGGAAACTAAAATG GCATTAAATTACAAAAGCAAATCACAGAAACGCTCTGCA GTAGAAAAGTCAATGAATACATTTTTGACCATTTATCTT 500 ATAATCCTTATTTCTGAAGCCATCATCAGTACTATCTTG AAATATACATGGCAAGCTGAAGAAAAATGGGATGAGCCT TGGTATAACCAAAAAACAGAACATCAAAGAAATAGTAGT AAGGTGGAATATGTGTTTACAGATAAAACGGGTACGCTG ACAG-3'

Figure 2.5. Characterization of PCR products with Electrophoresis and Sequence Analysis. Panel A, lane 1, Ikb ladder; lane 2, the single 742-bp genomic fragment from PCR with genomic DNA confirms RFBP is identical in size to the cDNA sequence. Panel B shows sequence obtained from genomic clones is identical to cDNA sequence. Underlined sequences denote PCR primer pair.

52 P type ATPases

Type I 1 Type V Heavy metal pumps Type II Type III No assigned H+, Mg2 + pumps Cu2+, cd2+ Ca 2+, Na+/K+and substrate specificity H+/K+ ATPases Type IV Phospholipid pumps T Yeast Nematode Bovinre Rabbit

class I DRS2 CEy49E10 ATPaseI class II YIE8 CEF36H2, CELF02C9 class III ATC5, YD8557.01 class IV YM8520.11 ? CELT 24H7 RFBP 9 CEW09D10

Figure 2.6. P-type ATPase Family. This classification of P-type ATPases is adapted from J. Mol Evol (1998) 46:84-101 and Genome Research (1998) 8:354-361. Type 1 ATPases include the heavy metal pumps while the non heavy metal pumps are distributed amongst Types II - V Names for pumps in yeast iSaccharomyces cerevisiae) and nematode (Caenorhabditis elegans) are shown. BLAST searches have identified RFBP as a member ofthe Type IV P-type ATPase family of phospholipid pumps. At the nucleotide level, RFBP has 58% identity with DRS2, and 53% identity with its bovine iBos taurus) ortholog (ATPase 11). RFBP has a higher identity (68%) with CELT24H7, which belongs to an as yet undefined class of Type IV P-type ATPases.

53 Coni'orniatiunally Flf.viblc Loop (Regions D-H)

Kej;ioas NH2 COOH

\ 7i Cytoplasm

1 2 3 5 6 7 8 9 10 Membrane 1 a uot ^ ,i^'

Figiu-e 2.7. Structure of a Classical P-type ATPase. Consensus sequences for the phosphorylation (P) site [DKTGT(L/I)T] and the ATP binding site (ATP) are located in the conformationally flexible loop. Transmembrane domains are numbered 1-10, and Region D is highlighted.

54 2.5 References Abel, K..I., Xu, .1., Yin, G.^'., Lyons, R.II., Meisler, M.Il. and Weber, B.L. (1995) Mouse Brcal: localization sequence analysis and identification of evolutionarily conserved domains. Hum Mol Gcnci, 4, 2265-2273.

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 25, 3389-3402.

Ambesi, A.. Pan, R.L. and Slayman, C.W. (1996) Alanine-scanning mutagenesis along membrane segment 4 ofthe \cast plasma membrane H+-ATPase. Effects on structure and function. J B/t>/ Chem, 271, 22999-23005.

.\ra\ind. L., Galperin, M.Y. and Koonin, E.V. (1998) The catalytic domain ofthe P-type .ATPase has the haloacid dehalogenase fold [see comments]. Trends Biochem Sci, 23, 127-129.

.Argos. P., Rao, J.K.. and Hargrave, P.A. (1982) Structural prediction of membrane-bound proteins. Eur J Biochem, 128, 565-575.

.\xelsen. K.B. and Palmgren, M.G. (1998) Evolution of substrate specificities in the P- type ATPase superfamily. J Mol Evol 46, 84-101.

Clarke. D.M., Loo, T.W., Inesi, G. and MacLennan, D.H. (1989) Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase. Nature, 339, 476-478.

Fan, CM. and Maniatis, T. (1990) A DNA-binding protein containing two widely separated zinc finger motifs that recognize the same DNA sequence. Genes Dev, 4. 29-42.

Freemont, P.S. (1993) The RING finger. A novel protein sequence motif related to the zinc finger. Ann N Y Acad Sci, 684, 174-192.

Geering, K. (2000) Topogenic motifs in P-type ATPases [In Process Citation]. JMembr Biol, 174, 181-190.

Hawkins, J.D. (1988) A survey on intron and exon lengths. Nucleic Acids Res, 16, 9893- 9908.

Hayward-Lester, A., Hewetson, A., Beale, E.G., Oefner, P.J., Doris, P.A. and Chilton, B.S. (1996) Cloning, characterization, and steroid-dependent posttranscriptional processing of RUSH-1 alpha and beta, two uteroglobin promoter-binding proteins. Mol Endocrinol, 10, 1335-1349.

55 HoiiM-as. \'., Bcne/.ra, M., Zhang, II., Manfrcdi, J.J., Weber, B.L. and Licht, J.D. (2000) BRC.M Plnsicallv and Functionally Interacts with AIT]. J Biol Chem 275 3(030-36:37.

.lensen, D.b.. Proctor, M., Marquis, S.T., Gardner, H.P., Ha, S.I., Chodosh, L.A., Ishov, A.M.. lommorup, N., Vissing, H., Sekido, Y., Minna, J., Borodovsky, A., Schultz, D.C.. W ilkinson, K.I),, Maul, G.G., Barlev, N., Berger, S.L., Prendergast, G.C and Rauscher, I J., 3rd. (1998) BAPl: a novel ubiquitin hydrolase which binds to the BRCAl RING finger and enhances BRCAl- mediated cell growth suppression. Oncogene, 16, 1097-1112.

Keller, .\.D. and Maniatis, T. (1991) Selection of sequences recognized by a DNA binding protein using a preparative southwestern blot. Nucleic Acids Res. 19, 4675-4680.

Kyte. J. and Doolittle, R.F (1982) A simple method for displaying the hydropathic character of a protein. J.\/o/ Biol, 157, 105-132.

Li, W.L, Chen, CL. and Chou, J.Y. (1989) Characterization of a temperature-sensitive beta-endorphin-secreting transformed endometrial cell line. Endocrinology, 125, 2862-2867.

Lingrel, J.B. and Kuntzweiler, T. (1994) Na+,K(+)-ATPase. J Biol Chem, 269, 19659- 19662.

Lovering, R.. Hanson, I.M., Borden, K.L., Martin, S., O'Reilly, N.J., Evan, G.I.. Rahman, D., Pappin, D.J., Trowsdale, J. and Freemont, P.S. (1993) Identification and preliminary characterization of a protein motif related to the zinc finger. Proc Null Acad Sci US A, 90, 2\\2-2\\6.

Lowrv', O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem.. 193, 265-175.

Lutsenko, S. and Kaplan, J.H. (1995) Organization of P-type ATPases: significance of structural diversity. Biochemistry, 34, 15607-15613.

Maegregor, P.F., Abate, C and Curran, T. (1990) Direct cloning of leucine zipper proteins: Jun binds cooperatively to the CRE with CRE-BPl. Oncogene, 5. 451- 458.

McKeown, M. (1992) Altemative mRNA splicing. Annu Rev Cell Biol, 8, 133-155.

Moller, J.v., Juul, B. and le Maire, M. (1996) Structural organization, ion transport, and energy transduction of P- type ATPases. Biochim Biophys .4cta, 1286, 1-51.

56 Pedersen, P.L. and Carafoli, E. (1987) Ion motive ATPases. I. Ubiquity, properties, and significance to cell function. Trends Biochem. Sci.

Scarborough, G..\. (1999) Structure and function ofthe P-type ATPases. Curr Opin Cell fi/W, 11,517-522.

Shaip, P.A. (1994) Split genes and RNA splicing. Cell, 77, 805-815.

Singh, H., Clcrc. R.G. and l.cBowitz, J.H. (1989) Molecular cloning of sequence-specific DNA binding proteins using recognition site probes. Biotechniques, 7, 252-261.

Singh, H.. LeBowitz, J.H., Baldwin, A.S., Jr. and Sharp, P.A. (1988) Molecular cloning of an enhancer binding protein: isolation by screening of an expression library with a recognition site DNA. Cell, 52, 415-423.

Smith, C.W., Patton, J.G. and Nadal-Ginard, B. (1989) Alternative splicing in the control of gene expression. Annu Rev Genet, 23, 527-577.

Thompson, J.D.. Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensiti\ity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Re^s, 22, 4673-4680.

Torii. K.U., Stoop-Myer, CD., Okamoto, H., Coleman, J.E., Matsui, M. and Deng, X.W. (1999) The RING finger motif of photomorphogenic repressor COPl specifically interacts with the R1NG-H2 motif of a novel Arabidopsis protein. J Biol Chem, 274.27674-27681.

Wu, L.C, Wang, Z.W., Tsan, J.T., Spillman, M.A., Phung, A., Xu, X.L., Yang, M.C, Hwang. L.Y., Bowcock, A.M. and Baer, R. (1996) Identification of a RING protein that can interact in vivo with the BRCAl gene product. Nat Genet, 14. 430-440.

57 CHAP lER III SUBCI'LLULAR LOCALIZATION OF RFBP

3.1 Introduction P-t\pe ATPases compri.sc a dixcrsc family of membrane transporters of eukar\otic and prokaryotic origin. These proteins are characterized by the formation of a co\alcntl> -bound aspartylphosphate intermediate, from ATP, to effect the transport of substrate across the membrane. There is a wealth of sequence data on P-type ATPases that has allowed the identification of core regions with characteristic signature sequences occurring in these proteins. Various members of this family of proteins have been the subject of se\ eral physiological and structural studies to unravel the functional and conformational properties that lead to ion transport. Members of this family are present in plasma membrane and organellar membranes in the cell. The NaVK^ ATPase, H^/K^ .ATPase and Ca'^ ATPase are all present in the plasma membrane. The sarcoendoplasmic reticulum Ca ^ ATPase (SERCA), as the name suggests, is present in the sarcoendoplasmic reticulum while the Cu^^ transporting ATPase, that has been implicated in Wilsons' disease in man (DiDonato and Sarkar, 1997), is present in the mitochondria (Lutsenko and Cooper, 1998). P-type ATPases that transport Cd^"^ have been identified in bacterial membranes too (Lutsenko and Kaplan, 1995). Type IV P-type ATPases are a recently identified class of membrane transporters. An ATP-dependent activity was initially implicated in the maintenance of asymmetric distribution of phosphatidylserine and phosphatidylcholine in chromaffin granules (Zachowski et al, 1989). Subsequendy this activity was isolated from erythrocytes (Morrot et al, 1990), chromaffin granules (Moriyama and Nelson, 1988) and synaptic vesicles (Hicks and Parsons, 1992). Tang et al (1996) eventually cloned the gene that codes for this ATPase from bovine chromaffin granules (ATPasell) and found it to be 67% similar to its yeast homolog, Drs2. Sequence analysis of ATPase II identified the DKTGTL/T motif that is characteristic of P-type ATPases. The presence of other signature motifs and transmembrane domains confirmed its structural identity as a P-type ATPase. Since a drs2 null mutant is unable to transport phosphatidylserine across the

58 plasma membrane, this suggests that these enzymes are members of a subfamily of P- l> pe A fPascs that transport phospholipids. This observation has however been challenged b\ Sicgmund el al. (1998) who did not detect a difference between wild type and drs2 mutant alleles in the transport of labeled lipid derivatives across the plasma membrane. W hile this ma\ mean that Dis2 and its orthologs are not phospholipid transporters, this ma> also suggest that this protein is not present at the plasma membrane in tlie first place and is therefore not required to maintain phospholipid asymmetry there. The latter h\pothesis has been validated by Chen el al. (1999) who did not detect any Drs2 at the plasma membrane by immunofluorescence or subcellular fractionation but in tact foimd Drs2 to be localized to late golgi membranes in yeast (Chen et al, 1999). It w as observed that a drs2 mutant exhibits late golgi defects that could possibly resuU from the loss of clathrin coat protein function. They propose that since the transbilayer movement of lipids can induce bending of membranes to facilitate vesicle budding, the Drs2 protein may participate in the formation of clathrin coated vesicles from the golgi membranes. On the other hand, an increase in the concentration of aminophospholipids on the cytoplasmic leaflet ofthe membranes may enhance the association of proteins on the golgi bodies to produce well-defined clathrin coated buds on its membrane. In order to gain better insight into the function of RFBP, which has been defined as a putative Type IV P-type ATPase by virtue of its sequence similarity to Drs2 and ATPasell, the goal of this study was to determine the subcellular location ofRFBP. This would also help us better understand its interaction with RUSH proteins and their contribution to the regulation of uteroglobin gene expression. In order to address this, initially Western blot analysis was carried out on subcellular fractions of endometrium from a hormonally manipulated female rabbit. This was followed by immunogold localization to visualize the nuclear location ofRFBP.

3.2 Materials and Methods 3.2.1 Reagents and Antibodies Rabbit antipeptide antibodies were made to a KLH-peptide at Research Genetics (Huntsville, AL). Amino acids 663-678 were selected because they displayed strong

59 antigenicit\ according to the PeptideStructure program (Genetics Computer Group Software, Madison, Wl), and because they are unique to RFBP. They share only 3 of 16 imiino acids common to any authentic I'ype IV P-type ATPase in the GenBank database. The antipeptide antibt)d\ titer was determined with an enzyme linked immunosorbent assa> with free peptide on the solid phase (1 pg/well), goat anti-rabbit IgG-horseradish peroxidase conjugate as the secondary antibody, and peroxidase dye. To reduce complications from non-specific binding, antibodies were affinity purified. Horseradish peroxidase-conjugated donkey anti-rabbit IgG was purchased from Amersham Pharmacia Biotech (Piscataway, New Jersey). Nitrocellulose transfer/immobilization membranes were purchased from Schleicher and Schuell (Keene, NH). Rainbow molecular weight markers and 10°o Tris-HCl gels were purchased from BioRad (Hercules, CA). Kodak X- OM.AT .\R film was purchased from Eastman Kodak Co. (Rochester, NY). Renaissance chemiluminescence reagents were purchased from NEN Life Science Products, Inc (Boston, MA). LR White resin and goat anti-rabbit IgG conjugated to 6nm or 15 nm gold were purchased from Electron Microscopy Sciences (Ft. Washington, PA).

3.2.2 Subcellular Fractionation of Endometrial Tissue Rabbit endometrium was homogenized (11 strokes) in 3 volumes (w/v) of buffer (10 mM Tris-HCl, pH 8.0; 100 mM KCI, 3 mM MgCL. 250 mM sucrose, and 5 mM dithiothreitol) using a Potter-Elvejhem motor driven Teflon pestle at 4°C. The whole tissue homogenate was fractionated by centrifugation/ultracentrifugation to obtain nuclear (2,000 x g). mitochondrial (10,000 x g), microsomal (165,000 x g) and postmicrosomal supernatant fractions for Western analysis. Detergent lysates (1% NP40) ofthe nuclear fraction were also prepared as a source of protein (22.5 pg/pl) in GST pulldown assays. The protein concentrations for all fractions were determined according to Dowry et a/. (1951) using BSA as the standard.

3.2.3 Polvacrvlamide Gel Electrophoresis and Western Blotting SDS-sample buffer was added to 40 pg aliquots of each isolated protein fraction. Samples were then heated at 65°C for 15 minutes and separated on a 10% native gel. Gels

60 were processed for Western analysis. Briefly, after transfer ofthe proteins to a nitrocellulose support, membranes were blocked in Tris-buffered saline (150 mM NaCl, 20 mM Tris-HCl, pi I 7.6) with 0.1% Tween 20 (TBST) and 2% powdered milk. Membranes were incubated o\crnight at 4°C in the same buffer containing affinity piuified anti-RFBP antibodies (1:50,000). Membranes were washed 3 x 30 min in TBST, and incubated in TBS 1 with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:5,000) for 90 min at room temperature. Membranes were subsequently washed 3 x 30 min in TBST. Specific signals were detected by chemiluminescence using Renaissance reagents.

3.2.4 Immuno Electron Microscopy Nuclei from endometrium of progesterone-treated rabbits were isolated + 0.1% Triton X-100 on sucrose-cushions containing protease inhibitors (Kleis-SanFrancisco et al. 1993). Nuclear pellets were resuspended in phosphate-buffered paraformaldehyde (4%). and fixed for 30 minutes. Nuclei were dehydrated in a graded series of ethanol and embedded in LR white resin (50°C). Thin sections (600-800 angstroms) were mounted on nickel grids, blocked with 5% normal goat serum (NGS) and incubated in antipeptide antibody (1:100 with 5% NGS) overnight at 4°C. Grids were rinsed and incubated with goat anti-rabbit IgG conjugated to 6 nm or 15 nm gold (1:25 with 5% NGS) for two hours at 37°C. Grids were fixed with phosphate-buffered glutaraldehyde (3%) for 5 minutes, stained with uranyl acetate and lead citrate and viewed with a Hitachi 600 electron microscope operated at 75 kV.

3.2.5 Animal Treatments All studies were conducted in accord with the NIH Guidelines for the Care and Use of Laboratory Animals as reviewed and approved by the Animal Care and Use Committee at Texas Tech University Health Sciences Center. Aduh New Zealand white rabbits (6 months of age) were housed for 3 wk before experimentation. Animals were injected every 24 hours with hormones and killed 24 hours after the last injection. Treatments (Chilton et al, 1988; Hayward-Lester et al, 1996; Kleis-SanFrancisco et al. 1993) included subcutaneous injections of progesterone (3 mg/kg/day for 5 days) and prolactin (2 mg/day for 5 days). Uterine endometrial tissue from these animals was then used for subcellular fractionation and for nuclear isolation.

3.3 Results 1 o rcsoUe samples by Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, SDS sample buffer was added to each sample and they were boiled for 5 minutes. Proteins were then resolved by electrophoresis and processed for Western anah sis. Upon de\ eloping with chemiluminescense reagents, little or no signal was detected in the lanes. Most ofthe signal was retained in the wells. This led to the conclusion that the protein was aggregating and therefore unable to enter the gel. The protocol was modified such that instead of boiling the samples, they were heated to 65°C before gel loading. This practice completely eliminated the putative aggregation phenomenon. Subsequently, analysis with affinity purified, antipeptide (IgG) antibodies prepared against RFBP amino acids 663-678 identified a 128-kDa immunoreactive protein in nuclear fractions from endometrium (Fig. 3.1). This observation indicated that RFBP is the first Type IV P-type ATPase to be located in the nuclear membrane. Based on the ClustalW alignment, the hydrophilicity plot, and the immunoanalysis data, RFBP would span the nuclear membrane 9 times leaving the NH2- and COOH-terminal ends on opposite sides ofthe bilayer. Each transmembrane domain is a 20-30 amino acid residue coiled into a single a-helix that approximates the thickness ofthe polar region ofthe lipid bilayer (Eisenberg et al, 1984). However, because the nuclear envelope is formed from two concentric membranes, it was not possible to deduce the membrane topology of RFBP from its primary structure alone. Because RFBP could be located in either the irmer or the outer nuclear membrane (Fig. 3.2), immunoelectron microscopy was used to show that RFBP is located in the inner nuclear membrane of nuclei isolated from rabbit endometrium (Fig. 3.3A). More specifically, immunogold labeling was used to show that the conformationally flexible loop extends into the nucleoplasm to contact euchromatin (Fig. 3.3B).

62 3.4 Discussion As mentioned earlier, all known type IV P-type ATPases are present in the plasma membrane or in golgi bodies. A striking feature ofRFBP, is its exclusive presence in the nuclear membrane. Nicotcra ct al. (1989) and Lanini cl al. (1992) have identified a nuclear 1\ pe II P-t> pe Ca'^-A'fPasc whose structure is that of a transporting pump. Howc\ er, no Type IV P-t> pe ATPases have been localized to the nuclear membrane. There are se\eral methods in use to define the topology of integral membrane proteins after theoretical anal\ sis has identified a-helical regions in the predicted secondary structure. These include laborious proteolytic digestions or chemical labeling with fluorescent site directed probes to determine membrane sidedness. The electron microscopic \isualization ofthe sidedness of gold labeled 2° antibody is an easy and direct approach if the epitope is not degraded during the process of sample preparafion. RFBP antibody is an anti-peptide antibody that is made to a unique region in the large hydrophilic loop ofRFBP. The disposidon ofthe loop in the cell and therefore the signal indicates the orientation ofthe protein in the nuclear membrane. As shown in Fig 3.4, the small hydrophilic loop (Regions A-C; 174 residues) extends into the perinuclear space that is contiguous with the lumen ofthe endoplasmic reticulum, and the large conformationally flexible loop (Regions E-H; 499 residues) extends into the nucleoplasm. Physical contact between the large loop and the euchromatin was confirmed with immunoelectron microscopy. A comparison (51-52) ofthe three- dimensional map ofthe neurospora plasma membrane H"^-ATPase (Auer et al, 1998) with the map ofthe sarcoendoplasmic reticulum Ca^^-ATPase shows their transmembrane domains are similar but their large loops differ substantially. The compact conformation of the Ca^"^-ATPase contrasts with the relatively open conformation ofthe large cytoplasmic loop ofthe H"^-ATPase, which extends to a maximum height of 80 angstroms above the membrane surface, and measures 85 x 50 angstroms in cross-section (Auer et al, 1998). Such conformational differences can be attributed to major rigid body interdomain movements (Kuhlbrandt et al, 1998).

The targeting of integral membrane proteins to the inner nuclear membrane is believed to occur by lateral diffusion along the endoplasmic reticulum and the nuclear

63 pore membrane domain followed by retention at the inner membrane. It will be interesting to determine the mechanism by which this Type IV P-type ATPase is targeted to and retained in the inner nuclear membrane ofthe cell.

64 12 3 4 5 215 kDa ^— ^

132 kDa

95 kDa

42.5 kDa

Figure 3.1. Western Analysis. Western blotting (40 pg protein/lane) was used to show the subcellular localization ofRFBP in endometrium. Antipeptide antibodies recognize RFBP (arrovk) in extracts of whole tissue (lane 1) and nuclear fractions (lane 2). RFBP expression was absent from mitochondrial (lane 3) and microsomal (lane 4) fractions, and barely detectable in the postmicrosomal supernatant (lane 5). Note rabbit IgG is recognized by the secondary antibody (donkey anti-rabbit IgG).

65 fi^'k . Cytoplasm T C P ,_ t P Outer N N Inner

Nucleus

Figure 3.2. Possible Topology ofRFBP. Immunological techniques have identified that RFBP is present in the nuclear membrane. This knowledge when combined with the sequence information gives the above possible orientations ofRFBP in this bilipid bilayer. Two other unlikely orientations, place the conformationally flexible loop in the perinuclear space. In order to determine whether RFBP is present in the outer or the inner nuclear bilayer, immunoelectron microscopy was performed with an antibody prepared against a highly antigenic region ofRFBP (*) in the conformationally flexible loop.

66 m

Figure 3.3. Immunolabeling of Ultra-thin Sections. Immunolabeling was initiated with affinit} purified, anti-RFBP antibodies, and goat anti-rabbit lgG/15 nm gold (Panel A) or goat anti-rabbit lgG/6 nm gold (Panel B). Immunolabeling (arrows) showed RFBP associated with the inner nuclear envelope (Panel A, 68,800x) and euchromatin (Panel B, 82,500x). Immunolabeling was excluded from nucleoli. Disrupted outer nuclear membranes (O) and intact inner nuclear membranes (1) are shown. Immunostaining was negligible in negative controls. When the outer membrane was selectively removed with Triton X-100, immunolabeling ofthe inner membrane and euchromatin was consistent with data depicted here.

67 Cytoplasm

Outer Membrane

j^u Regions Perinuclear V^ A-C Space

Inner 12 3 45678 9 Membrane Nucleoplasm COOH

% Conformationally Flexible Loop (Regions E-H)

^ *

Figure 3.4. Topology and Structure ofRFBP. Based on sequencing data and immunoelectron microscopy, the predicted topology ofRFBP is as shown in the figure. The small loop (Regions A-C) extends into the perinuclear space that is contiguous with the lumen ofthe endoplasmic reticulum, and the large conformafionally flexible loop (Regions E-H) extends into the nucleoplasm. Consensus sequences for the phosphorylation (P) site and the ATP binding site (ATP) are located in the conformationally flexible loop.

68 3.5 References

Auer, M.. Scarborough, G.A. and Kuhlbrandt, W. (1998) fhree-dimensional map ofthe plasma membrane ll+-ATPase in the open conformation. Nature, 392, 840-843.

Chen, C.^•.. Ingram, M.F., Rosal, P.H. and Graham, T.R. (1999) Role for Drs2p, a P-type ATPase and potential aminophospholipid translocasc, in yeast late Golgi funcfion. J Cell Biol, 147, 1223-1230.

Chilton, B.S., Mani. S.K. and Bullock, D.W. (1988) Servomechanism of prolactin and progesterone in regulating uterine gene expression. Mol Endocrinol, 2, 1169- 1175.

DiDonato, M. and Sarkar, B. (1997) Copper transport and its alterations in Menkes and Wilson diseases. Biochim Biophys Acta, 1360, 3-16.

Eisenberg, D., Schwarz, E., Komaromy, M. and Wall, R. (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol, 179, 125-142.

Hayward-Lester, A., Hewetson, A.. Beale, E.G., Oefner, P.J., Doris, P.A. and Chilton, B.S. (1996) Cloning, characterization, and steroid-dependent posttranscriptional processing of RUSH-1 alpha and beta, two uteroglobin promoter-binding proteins. Mol Endocrinol, 10, 1335-1349.

Hicks, B.W. and Parsons, S.M. (1992) Characterizafion ofthe P-type and V-type ATPases of cholinergic synaptic vesicles and coupling of nucleotide hydrolysis to acetylcholine transport. J Neurochem, 58, 1211-1220.

Kleis-SanFrancisco, S., Hewetson, A. and Chilton, B.S. (1993) Prolactin augments progesterone-dependent uteroglobin gene expression by modulating promoter- binding proteins. Mol Endocrinol, 7, 214-223.

Kuhlbrandt, W., Auer, M. and Scarborough, G.A. (1998) Structure ofthe P-type ATPases. Curr Opin Struct Biol, 8, 510-516.

Lanini, L., Bachs, O. and Carafoli, E. (1992) The calcium pump ofthe liver nuclear membrane is identical to that of endoplasmic reticulum. J Biol Chem, 267, 11548- 11552.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-175.

Lutsenko, S. and Cooper, M.J. (1998) Localization ofthe Wilson's disease protein product to mitochondria. Proc Nad Acad Sci USA, 95, 6004-6009.

69 Lutsenko. S. and Kaplan, J.H. (1995) Organization of P-type ATPases: significance of structural di\ersily. Biochcmislrv, 34, 15607-15613.

Mori>ama, ^' and Nelson, N. (1988) Purification and properties of a vanadate- and N- cth\ Imalcimide- .sensitive A'fPase from chromaffin granule membranes. J 5/'o/ (Vuw, 263, 8521-8527.

Morrot, G., Zachowski. A. and Dcvaux, P.F (1990) Partial purification and characterization ofthe human erythrocyte Mg2(+)-ATPase. A candidate aminophospholipid translocasc. FEBS Lett, 266, 29-32.

Nicotera, P.. McConkey. D.J., Jones, D.P and Orrenius, S. (1989) ATP stimulates Ca2+ uptake and increases the free Ca2+ concentration in isolated rat liver nuclei. Proc Null Acad Sci USA, 86, 453-457.

Siegmund, A., Grant, A., Angeletti, C, Malone, L., Nichols, J.W. and Rudolph, H.K. (1998) Loss of Drs2p does not abolish transfer of fluorescence-labeled phospholipids across the plasma membrane of Saccharomyces cerevisiae. J Biol Chem, 273, 34399-34405.

Tang, X., Halleck, M.S., Schlegel, R.A. and Williamson, P. (1996) A subfamily of P-type ATPase with aminophospholipid transporting activity. Science, 272,1495-1497.

Zachowski, A., Henry, J.P. and Devaux, P.F (1989) Control of transmembrane lipid asymmetry in chromaffin granules by an ATP-dependent protein. Nature, 340, 75-76.

70 CHAPTER IV IDENTIFICATION OF IHE RUSH BINDING SITE IN RFBP

4.1 Introduction Although se\ eral hundred proteins contain a RING finger motif establishing a function for this domain has been problematic because RING finger containing proteins are present in both the nuclear and cytoplasmic compartments ofthe cell and they participate in a range of biochemical processes in the cell. For example, the promyelocN tic leukemia oncoprotein (PML), that is a part of nuclear multiprotein complexes known as nuclear domains (NDIO), nuclear bodies or PML oncogenic domains (PODs), is involved in growth suppression (Mu et al, 1994), apoptosis (Hess and Korsme\er. 1998) and RNA transport (Lai and Borden, 2000). Another RING finger containing protein, KRAB-associated protein KAPl, is homologous to transcription intermediary factor ip (TIFIP) and is involved in transcriptional repression (Friedman et al. 1996; Peng et al, 2000). The RING finger containing protein c-Cbl is a cytosolic protein involved in the ubiquitination of receptor protein tyrosine kinases (Joazeiro et al, 1999). The common theme in all these proteins is that their RING domains participate in protein-protein interactions often resulting in the formation of multiprotein complexes (Borden, 2000). To understand the functional aspects ofthe interaction between RING finger proteins and their binding partners, it is critical to elucidate the molecular determinants of this interaction. There is extensive information available on the structural components of the RING domain that are likely to be critical to RING function. This information has mostly been garnered from the domain structures obtained by 'H-nuclear magnetic resonance for RING finger motifs of PML (Borden et al, 1995), equine herpes virus immediate early protein, lEEHV (Barlow et al, 1994), the human recombination protein, RAGl (Bellon et al, 1997) and more recently human MATl protein (Gervais et al, 2001). Database search for superimposable folds in the Protein Data Bank finds structural similarities between the RING motifs of MATl, RAGl and lEEHV proteins all of which adopt a p-P-a-P fold (Gervais et al, 2001). A slightly weaker structural homology is

71 obscrxcd with the RING domain of PML protein via its P-P-p-loop-a3,o-P fold (Borden ct al, 1995). All four structures bind two zinc atoms in a cross-brace arrangement and the zinc sites are separated by 14 angstroms, defining a conserved structural motif that is formed onl> in the presence of zinc (Fig. 1.3, Chapter 1). The motif is also characterized b> a conserxed h> drophobic core, from C4 to C5, and an overall positive surface charge w ith a few discrete negati\ e charges. This positive potential is the likely explanation for the non-specific binding of RING domains to DNA which is ten fold higher than for poly dIdC (Lo\ering ct al, 1993). The negative charges on the mofif on the other hand are essential to the function ofthe domain as a protein binding motif since mutafions in PML protein that alter the surface charge, without altering the ability ofthe motif to bind Zn2^ and fold, result in fewer nuclear bodies (Boddy et al, 1997). Aside from these common characteristics. RING domains are very different in their tertiary structures. This is due to \'aried spacing between Cysteines (C) C2-C3 and C5-C6. This spacing is very diverse both with respect to the primary sequence and the length. This gives rise to different three- dimensional folds reflecting the ability of different RING-containing proteins to bind different target molecules. Very little is known about the binding targets of RING domains. In an effort to understand the function ofthe RING domain, the search for RING finger binding partners has become quite intense. It has been observed that RINGs mostly bind other RJNGs and in \er\ few instances non-RING domains. For instance, the BRCAl RING-construct is able to homodimerize in solution and also form higher order oligomers. Yeast two hybrid analyses have independently identified three binding partners for the BRCAl RING finger, BRCAl associated protein-1, BAPl (Jensen et al, 1998), BRCAl associated RING domain 1, BARDl (Meza et al, 1999) and ATFl, a member ofthe cAMP response element-binding protein/activating transcription factor (CREB/ATF) family. While BAPl is a ubiquitin hydrolase, BARDl interacts with BRCAl via its own RING finger and together the two proteins are purported to participate in DNA repair. Like BAPl, ATFl is another non-RING containing binding partner for BRCAl. Its interactton with BRCAl may be physiologically relevant to the transcriptional activation of ATFl target genes, some of which are involved in the cellular response to DNA damage.

72 Previousl), onl> eight non-RING containing binding partners had been identified tor RING finger domains. Three of these proteins exhibit a proline-rich region of high similaritN (Borden, 2000; Kentsis and Borden, 2000). RFBP is the ninth protein that is a non-RING containing RlNCi binding protein. By visual inspection ofthe RFBP sequence, we were unable to identify a proline-rich region in RFBP suggesting that there is probabl) another domain specific to RFBP that mediates its binding to RUSH. The experiments discussed here were carried out (a) to confirm an in vivo interaction between RUSH and RFBP and (b) to begin to characterize the minimal domain ofRFBP that is recognized by the RUSH RING finger.

4.2 Materials and Methods 4.2.1 Reagents and Antibodies TaKaRa ExTaq enzyme and lOX LA PCR buffer were purchased from PanVera Corporation (Madison, WI). Sequenase Version 2.0 DNA Sequencing Kit, pGEX-2TK vector, glutathione sepharose 4B, protein A sepharose™CL-4B and isopropyl-p-D- thiogalactopyranoside (IPTG) were purchased from Amersham Pharmacia Biotech (Piscataway, New Jersey). BLR(DE3)pLysS cells were purchased from Novagen (Madison, WI). pcDNA3.1/Myc-His (-)B and the pCR™II vector which is a component ofthe TA Cloning Kit were purchased from Invitrogen (San Diego, CA). TNT®T7 Quick coupled Transcription/Translation System was purchased from Promega (Madison, WI). Kodak X-OMAT AR film was purchased from Eastman Kodak Co. (Rochester, NY). Renaissance chemiluminescence reagents were purchased from NEN Life Science Products, Inc (Boston, MA). Nitrocellulose transfer/immobilization membranes were purchased from Schleicher and Schuell (Keene, NH). TRAN^'S-label, 70% [^'S]- methionine, \5% [^^S]-cysteine was purchased from ICN (Irvine, CA). Rabbit antipeptide antibodies to RFBP and RUSH were made at Research Genetics (Huntsville, Alabama). Amino acids 663-678 ofRFBP were selected because they displayed strong antigenicity according to the PeptideStructure program (Genetics Computer Group Software, Madison, WI), and because they are unique to RFBP. They share only 3 of 16 amino acids common to any authentic Type IV P-type ATPase in the GenBank database.

73 Similarl), antipeptide antibodies were generated in rabbits against RUSH amino acids 370-3S7 (Hayward-Lester et al, 1996). The antipeptide antibody titer for both antibodies was determined with an en/yme linked immunosorbent assay with free peptide on the solid pha.sc (I pg/well), goal anti-rabbit IgG-horseradish peroxidase conjugate as the secondary antibod\, and peroxidase dye.

4.2.2 Preparation of Whole Cell and Nuclear Lysates Rabbit endometrium was homogenized (11 strokes) in 3 volumes (w/v) of buffer (10 mM Tris-HCl, pH 8.0; 100 mM KCI, 3 mM MgCL, 250 mM sucrose, and 5 mM dithiothreitol) using a Potter-Elvejhem motor driven Teflon pestle at 4°C. To test in vitro protein-protein interactions, NP40 was added to an aliquot ofthe whole tissue homogenate to a final concentration of 1%. This detergent lysate was re-homogenized (5 strokes), and centrifuged at 2000 x g to remove cellular debris. The supernatant fracfion was used as a source of protein (28.5 pg/pl) in co-immunoprecipitation and GST pulldown assays. The remaining whole tissue homogenate was fractionated by centrifugation/ultracentrifugation to obtain a nuclear (2000 x g) fraction also used in co- immunoprecipitation and GST pulldown assays.

4.2.3 Synthesis of GST-RING Fusion Protein The RING mofif (nucleotides 2433-2579; aa 755-803) was amplified from 50 ng of a 1030-bp partial RUSH cDNA clone as described above with a forward primer (5'- GCG^GATC^CG AAG AAT GTG CT-3') that had a unique BamHl site (bold) and a reverse primer (5'-GCG^AATT^CC ATG TAT ATC ATT-3') that had a unique EcoRI site (bold). To accommodate the low melting temperature of this primer pair, a four-step touch up PCR reacfion was performed with a hot start. The conditions were as follows: 30 s at 94°C, followed by 5 cycles of 94°C, 5 s; 35°C, 120 s; 5 cycles of 94°C, 5 s; 40°C 120 s; 5 cycles of 94°C, 5 s; 45°C, 120 s; 5 cycles of 94°C, 5 s; 50°C, 120 s; and a final extension for 10 min at 68°C. Samples were rapidly cooled to 4°C. A single 161-bp PCR product was cloned into pCR™II, excised with appropriate restriction enzymes and directionally subcloned into the BamHl-EcoRl sites ofthe pGEX-2TK vector. Insert

74 orientation was confirmed by sequencing in both directions by the dideoxy chain termination method. BLR(DI{3)pl,\ sS host cells were transformed with either the pGEX-2TK control or the p(il'\-2 IK-RlNG recombinant. Individual cultures (200-250 ml) were grown to an OD260 of 0.6, and IPTG was added to a final concentration of 1 mM. After 3 hours of additional growth, bacteria were pelleted by centrifugation at 5000 x g for 10 min. Bacteria were resuspended in I/IO''' volume of lysis buffer [50 mM Tris-HCl, pH 7.5, 100 mM NaCl, I niM EDTA, 0.5% NP40, leupepfin (1 pg/ml), anfipain (2 pg/ml), benzamidine (10 pg/ml), chymostatin (10 pg/ml), pepstafin (10 pg/ml), and phenylmethyl sulfonyl fluoride (2 mM)]. Bacteria were lysed on ice with mild sonication, and centrifuged at 10,000 x g for 20 min. Pellets were discarded and supematants were used directh (1 ml) for the pulldown assay using cell extracts or processed further to isolate ftision protein. To isolate the glutathione S-transferase (GST) and GST-RING fusion proteins, the supernatant was incubated with 1ml glutathione sepharose (50% v/v slurry in hsis buffer) for at least 60 minutes at 4°C. Beads were collected by centrifugatton at 500 X g for 5 minutes and washed three fimes with 10 bed volumes of ice-cold Ix phosphate-buffered saline (PBS; 140mM NaCl, 2.7 mM KCI, Na2HP04 and 1.8mM KH2PO4, pH 7.3). Sedimented beads were incubated with 0.5 ml elution buffer (10 mM glutathione in 50 mM Tris-HCl, pH 8.0) for 10 minutes to liberate either the GST or GST-RING fusion proteins. The samples were spun at 500 x g for 5 mins and the supernatant was collected. The elution step was repeated and the eluates were pooled and dialyzed overnight against 50 mM Tris-HCl pH 7.5. Lowry assay was carried out on the dialysates to estimate protein concentration, using BSA as a standard.

4.2.4 Synthesis ofRFBP Fusion Protein The hydrophilic loop region ofRFBP (nucleotides 1835-2413; amino acids 612- 804) was amplified from the original 1584-bp clone using primers with unique restriction enzyme sites (EcoR V and Hind HI, shown in bold). The forward primer 5"- GCGAT^ATCG GCC ATG GAC AGA CTG CAA GAT-3' was coupled with the reverse primer 5'-G CAA^GC^TTGC TCT AAT ATA ATA AAA ATG-3' in the

75 following PCR strategy: 30s at 94"C followed by 25 cycles of 5s at 94°C and 120s at 58.5 ^T. There was a final extension for 10 min at 68°C and samples were rapidly cooled to 4'^C. .\ 604-bp PCR product was subcloned into pCR™II, excised with EcoR V and Hind 111 and directionally subcloned into the corresponding sites ofthe pcDNA3.1/M:vc- His(-)B \ cctor. Insert orientation was confirmed by sequencing in both directions by the dideoxy chain termination method. In vitro transcription and translation ofthe construct was performed using the TNT-coupled rabbit reticulocyte 1\ sate according to the manufacturer's instructions. Briefl\. 1 pg DNA was added to the rabbit reticulocyte lysate containing T7 RNA poh merase, amino acid mix minus methionine and TRAN^^S-label. After 90 mins at 30°C, the protein products were resolved by electrophoresis on 10% SDS- gel to verify protein synthesis.

4.2.5 Co-immunoprecipitation and GST Pulldown using Cell Extracts or Recombinant RFBP For coimmunoprecipitation assays, aliquots of whole tissue homogenates were incubated overnight at 4°C with anti-RFBP, then incubated for a further 2 hours at 4°C with a 50% slurry of protein A-sepharose. Proteins were fractionated by SDS/PAGE in 10% gels and transferred to nitrocellulose membrane. Membranes were processed for Western analysis. Briefly, membranes were blocked in Tris-buffered saline (150 mM NaCl, 20 mM Tris-HCl, pH 7.6) with 0.1% Tween 20 (TBST) and 2% powdered milk. They were incubated overnight at 4°C in the same buffer containing affinity purified anti- RUSH (1:100; Hayward-Lester et al, 1996). The membranes were washed 3x30 min in TBST, and incubated in TBST with horseradish peroxidase-conjugated donkey anti- rabbit IgG (1:5,000) for 90 min at room temperature. Membranes were subsequently washed 3 x 30 min in TBST. Specific signals were detected by chemiluminescence using Renaissance reagents. For GST pulldown assays, GST and GST-RING containing supematants were mixed with whole cell or nuclear detergent lysates at 4°C for 90 min. Binding complexes were washed 3 x lysis buffer, fractionated by SDS/PAGE in 12.5% gels and transferred to

76 nitrocellulose membrane. Membranes were processed for Western analysis as described abo\c, except they were incubated with anti-RFBP antibodies (1:50,000). •fen micrograms of cither GST or GS T-RING protein were used in binding reactions containing 20 pi TNT reaction mix and 20 pi 50% v/v glutathione-sepharose sluiTN Tube contents were mixed at 4"C for 90 min. Bound complexes were washed 3x with Ix PBS. resuspended in 2 x SDS sample buffer and resolved by electrophoresis on 15%, SDS-gels.

4.3 Results RFBP was identified when an expression library was screened with the RING domain peptide to isolate a binding partner. In order to confirm the RING domain peptide w as capable of binding RFBP, GST pulldown assays were performed. The RING mofif from RUSH was expressed as a GST fusion protein (Fig. 4.1 A) and tested for its ability to bind specifically to native RFBP. Incubation of GST-RING with whole cell or nuclear extracts followed by recovery of bound RFBP on glutathion-Sepharose beads and immunoblotting with anti-RFBP confirmed the physical association of GST-RING and RFBP in vitro (Fig. 4.IB). In contrast, RFBP displayed negligible binding with GST alone (Fig. 4.IB). The in vivo interaction between RFBP and RUSH was verified by a companion experiment in which lysates from rabbit endometrium were subjected to immunoprecipitation with an antibody directed against RFBP, followed by immunoblotting with an anti-RUSH antibody. RUSH-la was identified by immunoblot analysis as a 113-kDa species present in RFBP immunoprecipitates (Fig. 4.2). The absence of RUSH-1 P (95-kDa) is explained by the fact that RUSH-la is the progesterone-dependent splice variant while RUSH-lp is the estrogen-dependent form (Hayward-Lester et al, 1996) and the tissue for these experiments was obtained from a progesterone treated animal. To ftirther define the subregion ofRFBP that binds to the RING finger, the coding sequence (1488 nucleofides, amino acids 612-1107) ofthe original RFBP clone was directionally subcloned into the EcoR V/Hind 111 sites ofthe eukaryotic expression \ector

77 pcDN.\3.lA\At-Ilis (-)B. Recombinant RFBP was labeled and evaluated by SDS/PAGE. llowe\er, recombinant RFBP has 6 transmembrane domains making it extremely hydrophobic and difficult to resohe by electrophoresis. Thus, the conformafionally flexible loop (Fig. 3.4, Chapter 111) of RFBP (amino acids 612-804) was directionally subcloned into the EcoR V'l lind III sites ofthe eukaryotic expression vector pcDNA3.1/.\/ic-His (-)B. Radiolabeled recombinant RFBP derived from this construct is -24 kDa and easily resolved by SDS-PAGE (Fig. 4.3A). GST-RING fusion protein c:apture d this in vitro transcribed/translated [^'S]-labeled RFBP (Fig. 4.3B). In future experiments, the protein sequence ofthe RFBP that retains RING finger binding ability (amino acids 612-804) will be subdivided and the GST-pulldown assays will be repeated. B\ reducing the target sequence by roughly one-half with each iteration, the search should converge rapidly to reveal a small segment ofRFBP sequence that is capable of binding to the RUSH RING finger.

4.4 Discussion RING-containing proteins are often present in multiprotein complexes and disruption ofthe RING motif results in disassembly of these complexes thus affecting biological ftinction. PML protein is present in speckled domains in the nucleus called nuclear bodies that are approximately 1pm in diameter and contain several proteins including SPIOO, Kr antigen and NDP52 (Lamond and Earnshaw, 1998). In vivo disruption of these domains, as observed in the acute promyelocytic leukemia (APL) pathological condition, results in the loss of PML nuclear bodies. In vitro disruption of these domains, as achieved by independently mutating two pairs of cysteines in the Zn^"^ binding sites of PML, aboUshes Zn^"^ binding (Borden et al, 1995). These studies suggest that a fully folded RING domain is necessary for PML to interact with other proteins and to form multiprotein complexes. Specificity in molecular recognition of RJNGs is demonstrated by the observation that the RING from Rptl, a putative lymphocyte specific transcription factor which has 90% sequence identity with the RING of BRCA is unable to support binding of BAPl when substituted for the BRCAl RING domain. Our library screening procedure for the

78 isolation ofRFBP and the co-immunoprecipitation experiments coupled with the GST pulldown from whole cell and nuclear lysates have conclusively demonstrated that there is a physical interaction between RllSI I and RFBP The interaction between a nuclear membrane protein with a transcription factor is an indication ofthe future of gene expression studies. Conventional approaches for understanding transcriptional regulation of target genes have been focused on the linear organization of promoter regulatory elements and identification and characterization of their cognate binding proteins. However, recent studies suggest that the study of transcription is no longer just limited these cis- and trans- elements as there is evidence to implicate components of nuclear structure in gene regulation even beyond the secondary organization of chromatin structure and nucleosome assembly (Workman and Kingston, 1998). This third level of organization includes contributions to gene expression b> components of nuclear architecture, more specifically proteins in the inner nuclear membrane and the nuclear matrix (Berezney et al, 1995; Spector, 1993). Therefore, it is biologically meaningful to investigate transcriptional activation and chromatin remodeling as interrelated processes within the context of nuclear architecture (Stein ero/., 1999; Wei era/., 1999). During the past decade the nuclear membrane has been the subject of intense scientific in\estigation due to its involvement in nuclear structure and dynamics during the cell cycle, chromatin attachment and organization, and its contribution to certain pathological states. The nuclear membrane is a bilipid bilayer. The outer nuclear membrane is continuous with the endoplasmic reticulum and the inner membrane lies directly between the outer nuclear membrane and the underlying filamentous nuclear lamina. At least three known proteins ofthe inner nuclear membrane (INM) physically interact with DNA-binding proteins (Goldberg et al, 1999). These include the Lamina associated polypepfide 2 (LAP2) (Dechat et al, 2000) and Emerin (Bione et al, 1994) both of which bind L2BP1 (LAP2 Binding Protein 1) (Furukawa, 1999) also called BAF (barrier to autointegration factor) (Lee and Craigie, 1998) identified in rat and mouse respecfively; and the Lamin B receptor (LBR) (Worman et al, 1988) that binds heterochromatin protein 1 (HPl) (Ye et al, 1997). BAFl is a small dimeric nuclear

79 protein that forms oligomeric complexes with DNA. It was discovered in retrovirus- infected cells where it promotes the ability of viral DNA to integrate into host DNA. While its normal function in cells is not known, it co-localizes with chromatin throughout interphase and mitosis and is the product of an essential gene that is conserved among eukaiy otes (Zheng ct al, 2000). LAP2 and emerin bind BAF through the LEM domain. I his domain is a 43-residue motif in the nucleoplasmic domain common to LAP2, emerin and MANI (Lin et al, 2000) all of which are residents ofthe INM. It has been suggested that BAF also binds M.ANl since the protein possesses the LEM domain. The affinity of B.AF for these proteins is yet to be determined although it has been speculated that this interaction is essential to nuclear organization as it mediates the anchoring of chromatin to the nuclear envelope.

Heterochromatin protein lor HPl is another small DNA-binding protein that was first identified in Drosophila where it is involved in position-effect variegation (Eissenberg et al, 1990). LBR association with HPl was identified in a yeast two-hybrid screen and it was further demonstrated that anti-LBR antibodies co-precipitate HPl from cell lysates (Ye et al, 1997). Binding between the two proteins may provide docking sites for heterochromatin at the nuclear periphery and modulate its higher order organization. The association of FNM proteins with chromatin or chromatin binding proteins may influence chromatin structure and thereby impact gene expression. Alternatively, the proper localization of DNA binding proteins like transcription factors and chromatin remodeling proteins at the nuclear envelope may depend on the binding of these regulatory factors to attachment sites provided by lamins or lamin binding proteins in the INM. RFBP is the first nuclear protein that interacts directly with a putative chromatin- remodeling partner. It is therefore tempting to speculate that RFBP could be part of a molecular mechanism that coordinates the spatial organization of regulatory proteins with RING domains within the organized nucleus. It will be important to determine if the localization and funcfional interaction ofRFBP with the domain in RUSH depends on lamins or lamin binding proteins and identify the functional basis of this interaction.

80 .\ : RING fusion protein

IP^ GST RING ^^ ^ GST

12 3 4 5 6 7 8

B : GST pulldown

215 kDa ^

132 kDa •• m 3, RFBP 95kDa>-

1 2

Figure 4.1. Synthesis of GST-RING Fusion Protein and GST Pulldown Assay using Whole tissue and Nuclear extracts. GST and GST-RING fusion proteins were synthesized in BLR(DE3)pLysS cells after inducfion with ImM IPTG (panel A, lanes 2,4,6,8). Protein products were immunoblotted and probed with anti-GST antibody. Uninduced controls are shown in lanes 1,3,5,7. The GST control is a 26 kDa protein product while the GST-RING fusion protein is -33 kDa. An in vitro pulldown assay with GST-RING followed by immunoblotting with the anti- RFBP antibody confirms the physical interaction between GST-RING and RFBP protein (Panel B). Dose-dependent binding is observed for GST-RING and RFBP from endometrial detergent lysates, i.e., lane 1(10 pl), lane 2 (20 pi), lane 3 (50 pi). Binding was negligible when the GST control was mixed with 20 pl ofthe same extract (lane 4). GST-RING binds RFBP (lane 5) in a nuclear detergent lysate (arrow). Molecular weight (MW) markers (kDa) are shown for each panel.

81 132 kDa

m 95 kDa RUSH

Figure 4.2. RUSH and RFBP Interact in vivo. Whole fissue detergent lysate was incubated with an antibody to RFBP. Western analysis ofthe immunoprecipitated lysates w ith an anti-RUSH antibody lights up a band at 113 kDa (arrow) confirming the physical interaction between RFBP and RUSH.

82 A : In vitro transcribed and translated RFBP fusion protein (amino acids 612-804)

RFBP fusion protein

B: GST pulldown

95 kDa

42.5 kDa •

19.5 kDa •• ~* ^ RFBP 612-804 7.5 kDa >

Figure 4.3. Synthesis ofRFBP Fusion Protein followed by a GST Pulldown using Recombinant GST-RING Fusion Protein and in vitro Synthesized Recombinant RFBP. RFBP nucleotides 1835-2413, corresponding to amino acids 612-804 were inserted into the expression vector pcDNA3.1/Mvc-His (-)B. A -22 kDa protein (panel A) was synthesized from this construct using the in vitro transcription /translation procedure. As shown in panel B, GST-RING fusion protein captured the in vitro transcribed/translated [^^S]-labeled RFBP (lane 1) while it did not bind the luciferase control (lane 2). Binding was negligible when the GST control was mixed with recombinant RFBP (lane 3) and luciferase (lane 4), Lower molecular mass species are degradation products or incomplete synthesis products from the in vitro translation reaction.

83 4.5 References Barlow, P.N.. Luisi, B., Milner, A., I-lliott, M. and Iwerett, R. (1994) Structure ofthe C31IC4 domain b\ Ill-nuclear magnetic resonance spectroscopy. A new structural class of zinc-finger. J Mol Biol, 237, 201-211.

Bellon, S.F., Rodgers. K.K.. Schatz, D.G., Coleman, J.E. and Steitz, T.A. (1997) Crystal structure ofthe RAGl dimerization domain reveals multiple zinc- binding motifs including a no\el zinc binuclear cluster. Nat Struct Biol, 4, 586-591.

Berezney, R, Mortillaro, M.J., Ma, H., Wei, X. and Samarabandu, J. (1995) The nuclear matrix: a structural milieu for genomic function. Int Rev Cytol, 1-65.

Bione. S., Maestrini, E., Rivella, S., Mancini, M., Regis, S., Romeo, G. and Toniolo, D. (1994) Identification of a novel X-linked gene responsible for Emery-Dreifliss muscular d\ strophy. Nal Gcnel, 8, 323-327.

Boddy, M.N., Duprez, E., Borden, K.L. and Freemont, P.S. (1997) Surface residue mutations ofthe PML RING finger domain alter the formation of nuclear matrix- associated PML bodies. JCe/Z^c/, 110, 2197-2205.

Borden, K.L. (2000) RING domains: master builders of molecular scaffolds? J A/o/ Biol, 295, 1103-1112.

Borden, K.L., Boddy, M.N., Lally, J., O'Reilly, N.J., Martin, S., Howe, K., Solomon, E. and Freemont, P.S. (1995) The solution structure ofthe RING finger domain from the acute promyelocytic leukaemia proto-oncoprotein PML. Embo J, 14, 1532- 1541.

Dechat, T.. Vlcek, S. and Foisner, R. (2000) Review: lamina-associated polypeptide 2 isoforms and related proteins in cell cycle-dependent nuclear structure dynamics. J Struct Biol, 129,335-345.

Eissenberg, J.C, James, T.C, Foster-Hartnett, D.M., Hartnett, T., Ngan, V and Elgin, S.C. (1990) Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster. Proc Nad Acad Sci USA, 87, 9923-9927.

Friedman, J.R., Fredericks, W.J., Jensen, D.E., Speicher. D.W., Huang, X.P., Neilson, E.G. and Rauscher, F.J., 3rd. (1996) KAP-1, a novel corepressor for the highly conserved KRAB repression domain. Genes Dev, 10, 2067-2078.

Furukawa, K. (1999) LAP2 binding protein 1 (L2BP1/BAF) is a candidate mediator of LAP2- chromafin interaction. J Ce//S'c/, 112, 2485-2492.

84 GeiAais, V., Busso, D., Wasielcwski, i;., Poterszman, A., Egly, J.-M., Thierry, J.-C and Kieffer, B. (2001) Solution structure ofthe N-terminal domain ofthe human FFllH MA f 1 subunit: new insights into the RING finger famWy. J Biol C 'hem, 276, 7457-7464.

Goldberg, M., Hard, A. and Ciruenbaum, Y. (1999) The nuclear lamina: molecular organization and interaction with chromatin. ('/// Rev Eukaryot Gene Expr, 9, 285-2^)3.

Hayward-Lester, A.. Hewetson. A., Beale, E.G., Oefner, P.J., Doris, P.A. and Chilton, B.S. (1996) Cloning, characterization, and steroid-dependent posttranscriptional processing of RUSFl-1 alpha and beta, two uteroglobin promoter-binding proteins. Mol Endocrinol, 10, 1335-1349.

Hess, J.L. and Korsmeyer, S.J. (1998) Life, death and nuclear spots [news; comment]. Nut Genci, 20, 220-222.

Jensen, D.E., Proctor, M., Marquis, S.T., Gardner. H.P., Ha, S.L, Chodosh, L.A., Ishov, .A.M., Tommerup, N., Vissing, H., Sekido, Y., Minna, J., Borodovsky, A., Schultz, D.C. Wilkinson, K.D., Maul, G.G., Barlev, N., Berger, S.L., Prendergast, G.C. and Rauscher, F.J., 3rd. (1998) BAPl: a novel ubiquitin hydrolase which binds to the BRCAl RING finger and enhances BRCAl- mediated cell growth suppression. Oncogene, 16, 1097-1112.

Joazeiro, C.A., Wing, S.S., Huang, H., Leverson, J.D., Hunter, T. and Liu, Y.C (1999) The tyrosine kinase negative regulator c-Cbl as a RING-type, E2- dependent ubiquitin-protein ligase [see comments]. Science, 286, 309-312.

Kentsis, A. and Borden, K.L.B. (2000) Construction of macromolecular assemblages in eukaryotic processes and their role in human disease: linking RINGs together. Current protein and peptide science, 1, 49-73.

Lai, H.K. and Borden, K.L. (2000) The promyelocytic leukemia (PML) protein suppresses cyclin Dl protein production by altering the nuclear cytoplasmic distribution of cyclin Dl mRNA. Oncogene, 19, 1623-1634.

Lamond, A.L and Earnshaw, W.C (1998) Structure and function in the nucleus. Science. 280, 547-553.

Lee, M.S. and Craigie, R. (1998) A previously unidentified host protein protects retroviral DNA from autointegration. Proc Nad Acad Sci USA, 95, 1528-1533.

85 I.in, F., Blake, D.L.. Callebaut, L, Skerjanc, I.S., llolmer, L., McBurney, M.W., Paulin- Levasseur, M. and Worman, H.J. (20()()) MANI, an inner nuclear membrane protein that shares the Ll'.M domain with lamina-associated polypeptide 2 and emerin../ Biol ('hem, 275, 4840-4847. lAnering. R., Hanson, I.M., Borden, K.L., Martin, S., O'Reilly, N.J., Evan, G.I., Rahman, D., Pappin, D.J., fiowsdale, J. and Freemont, P.S. (1993) Identification and preliminary characterization of a protein motif related to the zinc finger. Proc Null Acad.Sci T.S'.4, 90, 2112-2116.

Meza, J.E., Brzox ic, P.S.. King, M.C. and Klevit, R.E. (1999) Mapping the functional domains of BRCAl. Interaction ofthe ring finger domains of BRCAl and BARDl. J Biol Chem, 274, 5659-5665.

Mu, Z.M., Chin, K.V., Liu, J.H., Lozano, G. and Chang, K.S. (1994) PML, a growth suppressor disrupted in acute promyelocytic leukemia. Mol Cell Biol, 14, 6858- 6867.

Peng, H.. Begg, G.E., Schultz, D.C, Friedman, J.R., Jensen, D.E., Speicher, D.W. and Rauscher, F.J., 3rd. (2000) Reconstitution ofthe KRAB-KAP-1 repressor complex: a model system for defining the molecular anatomy of RING-B box- coiled-coil domain- mediated protein-protein interactions. J Mol Biol, 295, 1139- 1162.

Spector, D.L. (1993) Macromolecular domains within the cell nucleus. Annu Rev Cell Biol, 9, 265-315.

Workman, J.L. and Kingston, R.E. (1998) Alteration of nucleosome structure as a mechanism of transcriptional regulation. Annu Rev Biochem, 67, 545-579.

Worman, H.J., Yuan, J., Blobel, G. and Georgatos, S.D. (1988) A lamin B receptor in the nuclear envelope. Proc Nad Acad Sci USA, 85, 8531-8534.

Ye, Q., Callebaut, L, Pezhman, A., Courvalin, J.C. and Worman, H.J. (1997) Domain- specific interactions of human HPl-type chromodomain proteins and inner nuclear membrane protein LBR. J Biol Chem, 272, 14983-14989.

Zheng, R, Ghirlando, R., Lee, M.S., Mizuuchi, K., Krause, M. and Craigie, R. (2000) Barrier-to-autointegration factor (BAF) bridges DNA in a discrete, higher-order nucleoprotein complex. Proc Nad Acad Sci USA, 97, 8997-9002.

86 CHAPTER V QU AN riTATION OF RFBP IN 1 STROl IS AND HORMONALLY MANIPULAI I:D ENDOMiri RIUM USING COMPE 11 FIVE QUAN 111ATIVE RT-PCR

5.1 Introduction 5.1.1 Hormonal Regulation of P-type ATPases Links between hormone action, gene sequence, structure and function comprise one ofthe most compelling themes of molecular endocrinology. The FUNG-finger motif is a \er\ recent example of a sequence-structure family that is purported to mediate the formation of large protein complexes. It is the common feature of a superfamily of nearly 200 otherwise unrelated proteins, many of which are transcription factors, including the RL'SH family of chromatin remodeling proteins that bind to the Uteroglobin (UG) promoter. Previous chapters have described experiments that identify a RUSH binding partner in the inner nuclear membrane and authenticate this RUSH RING-finger binding partner, called RFBP, as a Type IV P-type ATPase. The potential for hormones to regulate the expression of P-type ATPases remains relatively unexplored. A few studies that have been carried out only consider the effects of hormones on the acfivity of P-type ATPases. For example, the demonstrafion that physiologically relevant concentrations of estradiol increased the hydrolytic activity of rat cortical Ca^"^ ATPase (Zylinska et al, 1999; Zylinska and Legutko, 1998) supports the idea that this plasma membrane calcium pump is a non-genomic steroid target (Zylinska et al, 1999). There are some experiments with the Menkes ATPase, which is a copper transporter implicated in human disorders of copper homeostasis, that suggest that hormones may affect the expression levels of ATPases. The protein is primarily localized to the trans-Golgi network showing a peri-nuclear staining pattern. Treatment of human breast carcinoma cells, PMC42, with a combination of estrogen, progesterone and prolactin, increased perinuclear (Golgi) and punctate (endosome) staining ofthe protein, as measiu-ed by indirect immunofluorescence (Ackland et al, 1997), even though

87 Northern anal\ sis failed to show whether this increase was due to increased mRNA s\nthesis.

.A recent stud> shows that the a isoform ofthe NaVK^-ATPase plays a critical role in sperm motility and fertilization (Woo cl al, 2000) implicating P-type ATPases in reproduction while simultaneously marking them as candidates for hormonal regulation. Studies have shown that RLlSll is hormonally regulated in the endometrium (Hayward- Lester et al, 1996; Robinson ci al, 1997). The a splice variant is increased in response to progesterone and prolactin while the p isoform is estrogen-dependent, ft is likely that RFBP, an inner membrane protein that binds RUSH, may be influenced by the hormonal environment too. It is known that hormones bind their receptors which are targeted to their binding sites in the promoters of hormonally responsive genes. Therefore hormones have a direct effect on gene expression. Hence, in order to determine if RFBP may be regulated b> hormones, I decided to use quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to measure RFBP expression levels in progesterone- and prolactin-treated female rabbits, knowing that these hormones modulate UG and RUSH expression.

5.1.2 Competitive RT-PCR A commonly used quantitative RT-PCR (Q-RT-PCR) procedure to measure transcript amounts is competitive quantitative RT-PCR. The technique involves the co- amplification of a known amount of a control template or internal standard within the experimental reactions to determine the amount of native or target message in the reaction. Previous attempts to quantify message using this system involved the use of a relatively invariant mRNA such as P-actin or another housekeeping gene as an internal standard. However, this type of multiplex PCR may be inaccurate because multiple sets of primers can interfere with the amplification of either or both ofthe native and internal standard templates thus influencing amplification efficiency. Additional sources of error are introduced when the expression ofthe internal standard actually varies. To minimize differences in the kinetics of primer annealing, which can lead to errors in quantitation, amplification ofthe internal control template must use the same

88 priiuer set that is used to amplify the natiye. Siebert and Larrick (1993) developed the commerciall) a\ailable PCR MIMIC sy.stem. This system employs a cDNA competitor template in the PCR step ofthe reaction. The advantage ofthe system is that it uses a heterologous competitor template to avoid the complication of heteroduplex formation between the native and competitor templates that can cause errors in quantification of reaction products. Howe\er this method does not control for differences in RT efficiencies for di\ erse templates that can affect number of template molecules available for the competitive PCR reaction. Therefore PCR MlMlCs have been modified to generate RNA MIMlCs that are then added as competitors to the reaction tube at the RT step such that both nati\ e and competitor templates go through the RT and PCR stages of the reaction in the same tube. This provides an accurate estimation ofthe number of nati\ e molecules in the reaction.

The aim ofthe present study was two-fold: (a) to develop and optimize the use of an RN.A MIMIC to evaluate RFBP message amounts in any given sample and then (b) use this system to measurem ofthe amount ofRFBP expression in the endometrium of hormonally manipulated rabbits. The expression ofRFBP, in reproductive and no- reprodutive tissues of male and female rabbits, was also evaluated using this system.

5.2 Materials and Methods 5.2.1 Reagents Sequenase Version 2.0 DNA Sequencing Kit and [a-^'S]dATP, 1000 Ci/mmol, for sequencing, were purchased from Amersham Pharmacia Biotech (Piscataway, New Jersey). TaKaRa ExTaq enzyme and lOX LA PCR buffer were purchased from PanVera Corporafion (Madison, Wl). The GeneAmp RNA PCR Core Kit and Amplitaq Gold DNA polymerase were purchased from Perkin Elmer Applied Biosystems (Foster City, CA). ). The pCRTi^ll and pCR®II-TOPO vectors are components of individual TA Cloning Kits that were purchased from Invitrogen (San Diego, CA). MetaPhor agarose was purchased from BioWhittaker Molecular Applications, Inc. (Rockland, ME). Large scale SP6 RNA transcription kit was purchased from Novagen (Madison, WI). The binary gradient HPLC system was purchased from Rainin Instrument Company, Inc.

89 (W oburn, M.A), and the DNASep columns containing alkylated polystyrene- di\ in> Ibenzene packing were purchased from 1 ransgenomic. Inc. (San Jose, CA). Tri Reagent vyas purchased from Molecular Research Center, Inc. (Cincinnati, OH).

5.2.2 .Animal Treatments .All studies were conducted in accord with the NIH Guidelines for the Care and Use of Laboratory Animals as reviewed and approved by the Animal Care and Use Committee at Texas Tech Uni\ersity Health Sciences Center. Adult New Zealand white rabbits (6 months of age) were housed for 3 wk before experimentation. Seventeen estrous animals were di\ ided into 2 groups. For group one, endometrium from six animals was pooled and RNA was isolated using a cold precipitation method (25). For group two. the rabbits were used in three experimental subgroups (n=3 animals/subgroup; 5 animals in the progesterone treatment category). Animals were injected every 24 hours w ith hormones and killed 24 hours after the last injection. Treatments (2, 24, 26) included subcutaneous injections of progesterone (3 mg/kg/day for 5 days), prolactin (2 mg/day for 5 days), or prolactin (5 days) followed by progesterone (5 days). For group 2 animals, total RNA was isolated from individual endometrial samples in Tri-Reagent according to Chomczynski and Sacchi (27). RNA concentrations for all samples were determined spectrophotometrically (A26o)- Electrophoretic fractionation and ethidium bromide staining confirmed the integrity of each sample.

5.2.3 Competitor Synthesis Clontech's MIMIC system was used to develop a heterologous competitor RNA (Fig 5.1). Competitor construction required two PCR amplification reactions. In the primary reaction, composite primers consisting of RFBP-specific sequence contiguous with 20-bp of MIMIC-specific sequence were designed to hybridize to opposite strands ofthe MIMIC DNA template, a 576-bp BamHl/EcoRl fragment ofthe v-erbB gene. For each 50 pl PCR reaction, MIMIC DNA template (2 ng) was mixed with TaKaRa ExTaq DNA polymerase (2 U/50 pl), TaqStart Antibody (0.44 pg/50 pl), deoxynucleoside triphosphates (0.2 mM each) and 0.4 pM of each composite primer. The MIMIC

90 sequence in each composite primer is underlined; forward, 5'-GTG CGT GGA CTC CCT AfG C IG FIT CCC AGC GCA AGT GAA ATC TCC ICC G-3- and reverse, 5'-GGA CiAG .AGT CAA CiAT GCT CC 1 (.TC GTf GGT TTC ATC TCC CTG TAT AAC A- 3'. .A total of 26 PCR cycles were performed for 45 seconds at 94^ C, 45 sec at 50° C, and 90 sec at 72^' C .Analysis of PCR products by agarose gel (1.5%) electrophoresis revealed a single 258-bp amplicon. A 1:100 dilution of this product was amplified again w ith gene-specific primers. This secondar) PCR reaction was performed in a 100 pl PCR reaction with TaKaRa ExTaq DNA polymerase (3 U/100 pl), TaqStart Antibody (0.66pg/100 pl), deoxN nucleoside triphosphates (0.2 mM each) and 0.4 pM of each RFBP-specific primer. The RFBP-specific primers were forward, 5'-GTG CGT GGA CTC CCT ATG CTG TTT CCC AG-3' and re\ erse, 5'-GGA GAG AGT CAA GAT GCT CCT GTC GTT GG-3'. A total of 18 PCR c\ cles were performed as described above. The quality and size ofthe final reaction product was again verified by gel electrophoresis. The PCR product was subcloned into pCR®ll-TOPO and closed circular DNA was purified by equilibrium centrifugation in CsCl-ethidium bromide gradients. The identity ofthe MIMIC competitor was confirmed by sequencing in both directions by the dideoxy chain termination method. The pCR®II-TOPO vector containing the MIMIC competitor cDNA insert was linearized with the restriction enzyme EcoRV and competitor RNA was transcribed using the standard SP6 RNA polymerase transcription protocol. RNA was precipitated with isopropanol to prevent the coprecipitation of free nucleotides resulting in a more accurate estimation ofthe RNA yield at 260 nm. RNA integrity was examined by denaturing agarose gel electrophoresis followed by visualization with ethidium bromide staining and UV illumination.

5.2.4 Competifive RT-PCR Serial dilutions of competitor RNA (6, 3, 1.5. 0.6, 0.3 and 0.1 pg) were made in yeast tRNA. These were combined with poly A+ RNA (1 ng) from HRE-H9 cells or total RNA (30 ng) from rabbit tissues, depending on the assay, and reverse transcribed with 91 MMLV and random hexamers for 30 minutes at 42" C. Products from this reaction were heated to 90^ c and cooled to 4" C Then 40 pl of a PCR mix containing Amplitaq Gold DN.A polymerase (1 U/50 pl) and 0.25 pM of each RFBP-specific primer described abo\ e were added to each reaction tube. PCR cycle parameters included a hotstart at 95'^'C for 2 minutes, 36 cycles of 45 seconds at 94'* C. 45 seconds at 67° C and 60 seconds at 72"^ C Samples were subjected to a final extension at 72° C for 5 minutes before rapid cooling to 4*^ C

5.2.5 HPLC Analysis of Reaction Products .Amplified products were quantified by means of a binary gradient HPLC system. Briefly, an aliquot (10 pl) of each RT-PCR reaction was injected onto a DNASep column containing an alkylated polystyrene-divinylbenzene packing and eluted in a 4-minute gradient of acetonitrile in 0.1 M triethylammonium acetate (pH 7.0) at a flow rate of 1 ml/min at room temperature. The amounts of native (166-bp) and compefitor (258-bp) products were determined by on-line UV absorbance detection at 254 nm.

5.2.6 Data Analysis An automated RT-PCR Microsoft plug-in program has been developed to facilitate data analysis ofthe peak areas for native and competitor products. The model estimates the area under each peak and translates that into transcript abundance from the ratio of reaction products and the amount of competitor added to the reaction. This model is available at http://www.grad.ttuhsc.edu/archive/index.html.

5.3 Results Primers were made to amplify a 166-bp region ofRFBP (3136-3301). The same primers were used in the construction of competitor RNA fragment that gave a 258-bp product. A titration analysis was carried out to determine if native and competitor templates demonstrated identical RT and PCR efficiencies. Briefly, varying amounts of competitor RNA (6, 3, 1.5, 0.6, 0.3, 0.1 pg) were mixed with Ing of poly A"" RNA from HRE-H9 cells, a rabbit endomett-ial cell line. Native and mutant products from the RT-PCR reaction were resolved

92 and quantified by denaturing IIPLC, a novel system used for the resolution and detection of vcr> low amounts of products (I la> ward-Lester el al, 1995). Fig. 5.2A shows chromatograms from one such titration analysis. A standard curve was also generated by plotting the log ratio of products on the >-a\is against the log competitor amount on the x-axis to calculate No or the amount of original native message present in the reaction. This analysis was repeated 5 times with consistent regression (R") and slope values close to 1 and -1 respectively. This mathematical anah sis displa> s that the system has identical efficiencies for the RT and PCR reaction steps ofthe experiment. A single dose of compefitor was then used to determine nati\e template concentration to determine if it gave a value similar to that obtained from the titration anah sis. .As shown in Fig. 5.3, the amount of native template determined from the single tube anal\ sis was similar to that determined from the titration analysis. The repeated single tube analysis (n = 10) of RNA from HRE-H9 cells yielded a coefficient of variation of 8.4° 0. This confirms the precision of the single-tube measurement with the denaturing HPLC quantification system. It also eliminates the need for titration analysis of each individual sample. Single tube analysis of total RNA extracted from the endometrium of hormonally treated rabbits shows that RFBP message is increased (p<0.01) in response to treatment with progesterone (Fig. 5.4). Prolactin plus progesterone further increased (p<0.01) the amount of message over the value for progesterone alone. The amount ofRFBP expressed corresponds to similar changes in the expression of RUSH, under these hormonal conditions. This suggests that there is tight regulation between the expression ofthe RING finger containing RUSH proteins and the RFBP. The single tube analysis was then used to determine the relative expression of native RFBP in male and female rabbit tissues by using a fixed concentration of competitor (1.1 pg) in an RT-PCR experiment. Quantification of competitive RT-PCR reactions by ion-pair reversed phase HPLC showed that RFBP message expression is ubiquitous and is present in varying amounts in all the tissues studied (Table 5.1). It is interesting to note that of all the reproductive tissues examined in the analysis, RFBP expression is maximal in the testis and ovary. These are also the same tissues that demonstrate abundant RUSH message expression.

93 5.4 Discussion Rae\maekers has mathematically expressed the relation between the amount of product obtained at the end ofthe PCR reaction with the efficiency ofthe reaction: N=No(l+E)" 5.1 where N is the amount of product at the end ofthe reaction, N„ is the initial amount of template, E is the efficiency ofthe reaction and n is the number of cycles. Therefore, for a competitive RT-PCR reacfion, mathematically,

T=T,(1+ET)" 5.2 C=C(l+Ec)" 5.3 where To and Co are the starting amount of native and competitor templates in the reaction tube; T and C are the amount of native and competitor at the end ofthe RT-PCR reaction after n number of cycles and E is the efficiency.

Therefore, log (T/C)=log To -log Co +ndog[(l+ ET)/(1+ Ec)]. If E r = Ec, then log (T/C)=log To -log Co. This is a linear equation ofthe form y=mx+c, 5.4 where y= log (T/C) and x= log Co; therefore the slope m is -1 and log To can be calculated by solving for a linear equation. The fitrafion analysis generates a standard curve with an R^ value of 0.99. This implies that the values lie along a straight line and one can use a linear equation to interpret the results. Therefore, according to the linear equation the slope is -1 which can only be true if the efficiencies ET and Ec are equal. RNA MIMICs have been used in the past but this is the first time that the system has been validated by mathematical analysis. Aside from a theoretical analysis, an experimental analysis can also be carried out by synthesizing native RNA and carrying out a reaction with known amount of native and competitor RNA and estimating the amount of native product. This can then be compared with the known value at the beginning ofthe reaction to calculate the efficiency ofthe reaction, and determine if the efficiencies ofthe native and competitor reactions are identical.

94 When this s\ stem was used to determine RFBP message levels in hormonally treated female rabbits, it was seen that message amount is increased (p<0.01) In response to treatment with progesterone while prolactin plus progesterone further increased (p<0.01) the amount of message o\cr the \alue for progesterone alone. The amount ofRFBP expressed corresponds to similar changes in the expression of RUSH, under these hormonal conditions. It is exciting to find that RFBP expression is hormonally regulated as this supports the speculation that there is tight regulation between the function of RUSH and RFBP, and further underscores the role ofRFBP as a facilitator of RUSH function in UG transcription.

95 t'oiii|iositc prinicr-s Miimc DNA

\

<^ AnriL-.!

I'liiiuirs I'C'R with compositL- primers

Ciene-specitV I'liiii.iiy PCR primers product

Anneal Secondary PCR with gene- specific primers

Purify secondary ^ TOPO Linearize and Competitor PCR product clone transcribe RNA

Figure 5.1. Oufline for the Construction of Competitor RNA from a PCR MIMIC Composite primers were used in the first PCR reaction, where as gene-specific primers were used in the second PCR reaction. Following size selection by agarose gel electrophoresis, PCR products from the second reaction were purified with the Geneclean II Kit (Bio 101. La Jolla, CA) and subcloned into the pCR®ll-TOPO vector. This template was linearized and transcribed. The resultant RNA was used as a competitor in the RT-PCR reactions.

96 LA JUL J 0=6 pg C=3pg C=1.5pg C=0.6 pg C=0.3 pg C=0.1 pg

\

\

\

— + - X \

log competitor amount

Figure 5.2. Validation ofthe Competitive RT-PCR system. Panel A: Chromatograms from a titration analysis. Aliquots of RT-PCR reaction products were injected onto an ion pair reversed-phase HPLC column. Amount of product was detected with on-line uv absorbance (254nm) and peak areas were quantified. Decreasing the amount of competitor (C) results in increasing synthesis of native (N) product. Panel B: Mathematical analysis. The log ratio of native and competitor products is plotted against the log of competitor input. The regression parameters (R =0.99, slope=0.98) indicate that the system meets the predicted theoretical ideals.

97 2000 --

Q. 1500 -- CD

o 1000 <

500 --

Titration Single Tube

Figure 5.3. Precision ofthe Single Tube Measurement. The amount of native template (y- axis) determined from the single tube analysis (n=10) was very close to the value determined by titration analysis (n=6). The repeated single tube analysis (n = 10) of RNA from HRE-H9 cells yielded a coefficient of variation of 8.4%.

98 1500 n 0 Estrous Control < Z CC W 1000 o H O) c o CO 500- Q. QQ cc

Estrous Estrous + P +PRL+P Hormonal Status

Figure 5.4. RFBP Expression is Hormone-dependent. Total RNA was extracted from the endometrium of hormonally manipulated does. The relative abundance ofRFBP in each sample (femtograms RFBP/30 nanograms total RNA) was calculated by HPLC analysis of native and competitor products from competitive RT-PCR reactions. Values are expressed as mean + SEM. Data were analyzed by ANOVA followed by Student- Newman-Keuls multiple range test (p < 0.05 significance level).

99 Fable 5.1. RFBP I'xpression is Ubiquitous. Total RNA was extracted from the tissues of adult male and female rabbits, l he relative abundance of RFBP in each tissue (femtograms RFBP/3() nanograms total RNA) was calculated b\ HPLC analysis of native and competitor products from competiti\ e RT-PCR reactions. Values arc expressed as mean ± SEM.

Tissue Male fissue Female

Bladder 1224 ± 136 Diaphragm 824 ± 88 Diaphragm 292 ± 50 Duodenum 1706 ± 18 Duodenum Heart 692 ± 138 Heart 425 ± 85 Kidne\ 1505 ± 115 Kidney 2394 ± 100 Liver 2091 ± 120 Liver 1351 ± 86 Lung 1185 ± 92 Lung 942 ± 47 Spleen 1454 ± 48 Spleen 986 ± 78 Testis 2301 ± 182 Ovary 1737 ± 171 Epididymis 1320 ± 220 Oviduct 672 ± 31 Vas 1093 ± 18 Uterus 683 ± 34 deferens Seminal 1218 ± 205 Cervix 1356 ± 33 vesicle Prostate 1338 ± 23 Vagina 818 ± 28

100 5.5 References .Ackland, M.L., Cornish, E.J., Paynter, J.A., Grimes, A., Michalczyk, A. and Mercer, J.F (1997) Expression of Menkes disease gene in mammary carcinoma cells. Biochem ,/, 328, 237-243. lla\ ward-Lester, .A., llewet.son. A., Beale, E.G., Oefner, P.J., Doris, P.A. and Chilton, B.S. (1^)^)6) Cloning, characterization, and steroid-dependent posttranscriptional processing of RUSll-1 alpha and beta, two uteroglobin promoter-binding proteins. Mol Endocrinol, 10, 1335-1349.

Hayward-Lester, .A., Oefner, P.J., Sabatini, S. and Doris, P.A. (1995) Accurate and absolute quantitative measurement of gene expression by single-tube RT-PCR and HPLC. Genome Res, 5, 494-499.

Robinson, C..\.. Hayward-Lester, A., Hewetson, A., Oefner. P.J., Doris, P.A. and Chilton, B.S. (1997) Quantification of alternatively spliced RUSH mRNA isoforms by QRT-PCR and IP-RP-HPLC analysis: a new approach to measuring regulated splicing efficiency. Gene, 198, 1-4.

Siebert. P.D. and Larrick, J.W. (1993) PCR MIMICS: Cometitive DNA fragments for use as internal standards in quantitative PCR. BioTechniques, 14, 244-249.

Woo, A.L., James, P.F. and Lingrel, J.B. (2000) Sperm motility is dependent on a unique isoform ofthe Na,K-ATPase. J Biol Chem, 275, 20693-20699.

Z\ linska, L., Gromadzinska, E. and Lachowicz, L. (1999) Short-time effects of neuroactive steroids on rat cortical Ca2+-ATPase activity. Biochim Biophys Acta, 1437. 257-264.

Zylinska, L. and Legutko, B. (1998) Neuroactive steroids modulate in vitro the Mg(2+)- dependent Ca(2-H)- ATPase activity in cultured rat neurons. Gen Pharmacol, 30, 533-536.

101 CHAP f ER VI CONCLUSIONS AND DISCUSSION

6.1 Summary of Results lukaryotic transcription is an intricate biochemical process that is tightly regulated at several levels. There are an astounding number of DNA-binding proteins and regulator) factors that work coordinately to initiate and sustain the event. The RUSH proteins belong to such a family of DNA-binding proteins. They bind to the upstream promoter region ofthe uteroglobin (UG) gene in the rabbit endometrium (Hayward- Lester ct al, 1996; Kleis-SanFrancisco et al, 1993); the B-box of plasminogen activator inhibitor gene-1 in HeLa cells and the human skeletal muscle (Ding et al, 1996) and tumor necrosis factor-response element in a mouse preadipocyte cell line (Zhang et al, 1997). Our focus in the lab has been the binding of RUSH to the upstream promoter region ofthe UG gene in the rabbit endometrium. RUSH proteins contain seven motifs that are characteristic ofthe SWI/SNF family of chromatin remodeling proteins (Hayward-Lester et al, 1996). They also have a recently characterized motif called the RJNG-finger (Borden and Freemont, 1996; Freemont, 1993; Freemont et al, 1991). This mofif is present in over 200 proteins, many of which are transcription factors. The objective ofthe work contained in this thesis was to identify a protein that binds to the RUSH protein via its RING finger domain and provide insight into the characteristics of this binding partner, that would shed light on its interaction with RUSH in regulating UG gene transcription. The rationale for looking for a protein that binds the RING-finger domain ofthe RUSH proteins is based on the evidence in the literature implicating the RING-finger domain in mediating protein-protein interactions (Borden, 2000; Saurin et al, 1996). Therefore, an expression library was screened with the RUSH RING finger in order to isolate a binding partner for this protein. Chapter II describes the library screening procedure used to isolate the clone for this protein that has been christened the RING Finger Binding Protein or RFBP. RACE PCR was carried out with cDNA libraries made from rabbit endometrial poly A^ RNA, to extend the original clone that was 1584-bp long. The composite sequence (4286-bp) lacks

102 an initiation signal but contains a stop codon (TAA) and a canonical polyadenylation signal with a poly .\ tail. BL.AST searches of databases with the RFBP cDNA sequence identified RFBP as a member ofthe P-type A'lPase family of integral membrane transport proteins (Moller cl al, 1996), more specifically the subclass of Type IV P-type ATPases (.\xelsen and Palmgren, 1998; Tang el al, 1996). ClustalW alignment ofthe predicted sequence w ith other known I ype IV P-type ATPases identified seven of eight signature sequences characteristic of this class of proteins and nine transmembrane domains. The putati\ e transmembrane domains were also identified by hydrophilicity analysis using the Kyte-Doolittle algorithm (Kyte and Doolittle, 1982) along with the .Vrgos et al. (1982). transmembrane analysis, the Tmpred program (www.ch.embnet.org) and the SOSUI system (http://azusa.proteome.bio.tuat.ac.jp/sosui/).

The next objective was to identify the cellular compartment where RFBP is localized. Chapter 111 describes the subcellular fractionation method used to isolate nuclear, mitochondrial and microsomal and post microsomal fractions that constitute the biochemical compartments obtained upon centrifugafion. These fractions were subjected to Western analysis using an anti-peptide antibody raised to RFBP. It was determined that RFBP is present in the nuclear compartment in the cell. This is an exciting finding as it is the first biochemical evidence of a Type IV P-type ATPase being localized to the nucleus. However, the nuclear membrane is a bilipid bilayer and since RFBP is an integral membrane protein, it became imperative to determine whether it is present in the outer or the inner nuclear membrane. Therefore immunoelectron microscopy was used to ascertain that RFBP is present in the inner nuclear membrane with its conformationally flexible loop oriented in the nucleoplasm. RFBP was isolated from a library screen using the RING finger motif of RUSH. However, do the two proteins interact in vivo? What regions of these proteins are involved in this binding? These questions gave rise to the experiments discussed in Chapter IV Co-immunoprecipitation experiments carried out with the antibody to RFBP brought down the RUSH protein from a whole cell extract suggesting that the two proteins bind each other in the cell. At the same time, a RING-GST fusion protein was synthesized and a GST pulldown assay was carried out with a RUSH RING-GST

103 construct. .\s expected, RF BP was pulled down from a whole cell and a nuclear extract turther confirming that the two proteins physically interact in the cell. The next part of the experiment was to determine die minimal sequence in RFBP that binds the RUSH RING finger, fhe expression library screen pulled out a 1584-bp clone that extends from aa 612-1107 ofthe current 4286-bp (496 aa) clone. Therefore, this clone is likely to contain the RING binding domain. The original strategy was to use the coding sequence (1488-bp) of this clone to carry out the binding assay. However, recombinant RFBP has 6 transmembrane domains making it extremely hydrophobic and difficult to resolve by electrophoresis. As a result, only the hydrophilic region ofthe original RFBP clone (amino acids 612-804) was subcloned into an expression vector and an RFBP fusion protein was s> nthesized in a TNT reaction. Radiolabeled recombinant RFBP bound immobilized GST-RING idenfifying a RING binding domain between aa 612-804 ofthe RFBP sequence. This region is 193 aa long and the ultimate objective is to further characterize this region to determine the minimal RING binding domain in the predicted RFBP sequence. In order to look at the abundance ofRFBP message in the endometrium in the progesterone ± prolactin milieu, which is known to affect RUSH message levels (Hayward-Lester et al, 1996), a quantitative competitive RT-PCR analysis was carried out. A heterologous competitor RNA was synthesized and used in conjunction with a competitive RT-PCR strategy to quantify native messenger RNA levels. Once validated, this system was employed to quantify the message levels under different hormonal conditions. These results, discussed in Chapter V, demonstrate that the message for RFBP was significantly increased in response to progesterone (p<0.01) and was increased further still in the presence of both progesterone and prolactin (p<0.01) indicating that RFBP message levels are hormonally regulated. The quantitative RT-PCR system was also used to examine the abundance of RFBP message in different tissues in both male and female rabbits. As presented in Chapter V, RFBP expression varies in the different tissues examined and is particularly high in liver, kidney and the reproductive tissues testis and ovary. These are also the same tissues that demonstrate abundant RUSH expression (Robinson et al, 1997).

104 6.2 Discussion fhcse studies clearly establish that RFBP is a novel protein ofthe inner nuclear membrane. The unique localization of this Type IV P-type ATPase and its physical association with RUSH transcription factors provides us with a clue into its possible functional role. Known proteins ofthe inner nuclear membrane like the Lamin B receptor (LBR) (Worman el al, 1988), lamina associated polypeptides (LAP)-land 2 (Furukawa Cl al, 1995; Martin cl al, 1995) and emerin (Bione et al, 1994) are already being extensi\ely studied for their involvement in the functional organization ofthe nucleus. This leads us to speculate that, like other residents ofthe inner nuclear membrane, RFBP also pla\ s a role in the organization ofthe nucleus. It may do so by facilitating the localization of RING finger containing transcription factors to subnuclear domains that support gene expression. So what is functional organization ofthe nucleus and how may RFBP be a part of this organization? Functional organization refers to the higher order structure prevalent in the nucleus that maintains biochemical processes like DNA replication, transcription and RNA processing in discrete domains. The presence of these functional domains has been clearly demonstrated by elegant experiments conducted by Ronald Berezney and his group, and Peter Cook and his colleagues, who visualized distinct transcription and replication factories that are arranged in domains (Cook, 1999; Jackson et al, 1993; Wei et al, 1999). Each domain contains numerous sites, with three-dimensional network-like arrays (Jackson et al. 1998), emiched in regulatory factors like RNA polymerase II subunits, nascent transcripts. Proximal sequence element (PSE)-binding transcription factor (PTF) and Octl(Pombo and Cook, 1996; Pombo et al, 1998). This domain organization is present even after the removal of chromatin suggesting that chromatin itself is not the fiandamental structure organizing the nucleus. In fact the spatial and temporal association of these domains with a nuclear fraction called the nuclear matrix (Ma et al, 1999; Nakayasu and Berezney, 1989) has led to the view that the matrix may provide the three-dimensional scaffold thus integrating nuclear architecture with nuclear function.

105 The nuclear matrix was first described by Russian investigators Zbarskii and DeboN (Zbarskii and Debov, 1948) as a residual structure resulting from the extraction of nuclei w ith a high .salt solution. Later in 1974, Berezney and Coffey coined the term nuclear protein matrix (Berezney and Coffey, 1974) for this nuclear fraction that they discovered was 98.4"o protein. It has since been demonstrated to be a proteinaceous network of filaments that is present throughout the volume ofthe cell (Nickerson et al, 1995, 1997) and appears to be the nuclear equivalent ofthe cytoskeleton that provides structure to the cell. Aside from the replication and transcription factories observed by earlier scientists, recent immunofluorescence and electron microscopic imaging procedures have identified numerous small sites rich in serine- and arginine-rich proteins called SR proteins (Iborra et al, 1998), nuclear bodies (Ascoli and Maul, 1991; Dyck et al. 1994)and coiled bodies (Gall et al, 1995) that present a speckled pattern in the nucleus. These sites are putatively involved in transcription and RNA processing and trafficking (Carter et al, 1993; Doucas, 2000; Misteli et al, 1997). These macromolecular assemblies are also associated with the nuclear matrix (Dyck et al, 1994) providing further evidence that the filamentous nucleoskeleton provides the structural framework for organizing factors in the nucleus. The significance of nuclear structure to gene expression is further highlighted by the following key observations: i. Actively transcribing genes are associated with the nuclear matrix. These genes bind to the nuclear matrix by approximately 200-bp long A/T-rich Matrix/Scaffold associated regions (M/SARs) present in the enhancer or intronic sequences ofthe genes (Stief e/ al, 1989). ii. Experiments to identify regulatory factors that associate with the nuclear matrix have identified several nuclear proteins. These include type II and type III RNA polymerase subunits (Vincent et al, 1996), type II DNA topoisomerase (Berrios et al, 1985), histone deacytelase (Hendzel et al, 1991), steroid hormone receptors (Barrack, 1987) and other DNA binding factors that bind to MARs or SARs. iii. Some ofthe transcription factors identified in the nuclear matrix fraction are common to all cell types and physiological states while a significant number of them appear to

106 be cell- and tissue- type specific. SP-1, ATF, OCT-1 and API are some ofthe transcription factors that can be detected in matrix and non-matrix fractions in several cell t> pes (\ an W ijnen ci al, 1993). However C/EBP proteins and the NMP-2, which also recognizes binding sites resembling the consensus CCAAT sequence for C/EBP, are cell-l\ pe specific factors involved in osteocalcin gene expression and are restricted exclusively to the nuclear matrix of specific cell types (Bidwell el al, 1993; van W ijnen et al, 1993). The retinoblastoma protein (Rb) associates with the nuclear matrix in a cell cycle-dependent manner. This association is influenced by the phosphor) lation status ofthe protein (Mancini el al, 1994; Mittnacht and Weinberg, 1991). These obserxations clearly indicate that the nuclear matrix plays a role in the tissue specific regulation of gene expression. i\ . Investigators have also identified components ofthe nuclear matrix that may participate in the binding and specific localization of transcription factors to the matrix. The progesterone receptor binds to the receptor binding factor-1 (RBF-1) w hich is a resident protein of the nuclear matrix that has been identified as an acceptor protein for activated progesterone receptor (Schuchard et al, 1991). Thus protein associations with the nuclear matrix that localize and sequester regulatory factors coupled with the presence of transcription competent genes also in the nuclear matrix are indicative of primary organizational roles ofthe nuclear matrix. This has given rise to the current hypothesis that the nuclear matrix acts as a scaffold by facilitating the specific interactions of transcription factors with DNA sequences (Landers and Spelsberg, 1992). In order to understand how the nuclear matrix may participate in organizing transcriptional events, it has become necessary to identify the basic molecular components ofthe nuclear infrastructure. The structural framework for the nuclear matrix is provided by branched 10 nm filaments present throughout the nucleus (Hendzel et al, 1999- Nickerson et al, 1997) interacting with the peripheral nuclear lamina. (Gruenbaum et al, 2000). Actin (Nakayasu and Ueda, 1985) and some as yet unidentified ribonucleoproteins have also been localized to the matrix and together these proteins are suggested to constitute the nucleoskeleton.

107 face ofthe inner nuclear membrane. Lamins together w ith kunin binding proteins, present in the inner nuclear membrane, constitute the nuclear lamina structure. Lamins are classified into two types, A and B, that differ in their amino acid sequence, expression and localization patterns (Stuurman el al, 1998). Differential splicing ofthe lamin A gene gives rise to lamin C (Fisher et al, 1986). Structuralh . all lamins are composed of an a-helical rod domain flanked by a short head at the amino terminal and a long carboxy-terminal tail. While B-type lamins are always membrane associated, lamins A and C are also found in the cell interior. An interesting difference between the two lamin types is that B-type lamins are present in all cell types, but the .A-t\pe lamins, including lamin A and C, are only present in differentiated cell t>pes. In vivo, lamins are closely associated with chromatin fibers (Belmont et al, 1993) and under in vitro conditions both A and B type lamins can bind histones (Taniura et al, 1995), mitotic chromosomes (Glass et al, 1993) and also specific MAR sequences (Zhao et al, 1996). The chromatin interacting region has been localized to the tail domain ofthe protein. While these proteins are postulated to play structural roles (Dechat et al, 1998). their interactions with chromatin and histones provide strong evidence that lamins are directh involved in influencing the localization of chromosomes or genes to the nuclear matrix.

6.2.2 Lamin-binding Proteins Lamins bind integral and peripheral membrane proteins ofthe inner nuclear membrane. These include the lamina associated polypeptide (LAP)-l and 2(3, the lamin binding receptor (LBR), emerin and MANI (Paulin-Levasseur et al, 1996), to name a few. These proteins are fiindamentally involved in the assembly of lamin structures and due to their observed interactions with lamins, chromatin and DNA-binding proteins (Gotzmann and Foisner, 1999) are suggested to aid in the assembly of higher order chromatin domains. All these proteins may play a coordinated role in nuclear architectural control of gene expression.

108 6JJLActin There is a large body of literature providing evidence for the localizafion of actin in the nucleus. These include both biochemical and ultrastructural studies that demonstrate that filamentous actin is localized to the nuclear matrix (Amankwah and De Boni, 1994; Nakayasu and Ueda, 1983). Since an in vitro interaction between actin and the carbox) 1 terminal domain of lamin A has been observed (Sasseville and Langelier, 1998), it could be implicated along with lamins in the organization of functional chromatin domains. There is mounting evidence for the presence of several actin-related proteins (.VRPs), Arpl through ArplO (Boyer and Peterson, 2000) in the nucleus of se\eral organisms and their involvement in chromatin remodeling. Purification ofthe SWI SNF like Brg-asssociated factor (BAF) complex from T-lymphocytes has allowed the identification of P-actin as one ofthe subunits ofthe complex along with a novel ARP called B.AF53 that is homologous to Arp3 (Zhao et al, 1998). A BAF53 ortholog has also been identified in Drosophila in association with the Drosophila equivalent ofthe SWI/SNF complex called the Brm associated complex (BAP) (Papoulas et al, 1998). Howe\ er this protein seems to be absent in yeast which instead possesses Arp 7 and 9 in association with the SWI/SNF complex (Peterson et al, 1998). Studies have begun to elucidate the role of these proteins that are intimately associated with the chromatin remodeling machinery ofthe cell since their knockouts demonstrate the .sw/A/7/"mutant phenotype. Mutating the ATP binding and hydrolysis domains does not have any effect on SWJ/SNF activity. This is interesting because the mammalian BAF complex was found to be associated with the nuclear matrix while a complex lacking the actin and BAF53 subunits fails to localize to the nuclear matrix (Zhao et al, 1998). Thus actin and its related proteins might have a direct role in reorganizing chromatin at the nuclear matrix.

6.3 Future Studies Having established the fundamental details regarding the functional organization ofthe nucleus and the molecular components of nuclear architecture that mediate this organization, one can begin to investigate the role ofRFBP, a novel member ofthe inner

109 nuclear membrane that binds the RUSH transcription lactors, in this organization. PrimaiA questions to ask would be whether RFBP i. co-locali/es with RUSH, ii. interacts w ith the nuclear matrix, iii. associates with lamins, 'i\. is responsible for the discrete affiliation of chromatin-remodeling machines, via their RlNG-domains, with transcriptionally active genes, \. is affiliated w ith transcription factories or RNA processing centers/splicing complexes, vi. is the subnuclear distribufion ofRFBP under hormonal control? Answ ers to these questions will not only provide insight into the association of RFBP with the RUSH proteins and the consequent impact on UG gene regulation, they will also shed light on the contribution ofRFBP to nuclear events like transcription in the context of structural elements ofthe nucleus. With the emerging discovery of proteins that are present in the nuclear envelope, studies on the nuclear architectural control of gene expression are at an undoubtedly exciting phase. RFBP is one ofthe few proteins identified to date that is present in the inner nuclear membrane and its interaction with a transcription factor suggests that it may be a link between nuclear structure and transcription. The work contained in this thesis is just the tip ofthe iceberg providing the basis for further studies on mechanisms of gene transcription and regulation.

10 6.4 References Amankwah, K.S. and De Boni, U. (1994) Ultrastructural localization of filamentous actin within neuronal interphase nuclei in situ. Exp Cell Res, 210, 315-325.

.\rgos. P., Rao, J.K. and Hargra\e. P.A. (1982) Structural prediction of membrane-bound proteins. Eur J Biochem, 128, 565-575.

Ascoli, CA. and Maul. Q^.G. (1991) Identification of a novel nuclear domain. JCW/ Biol, 112,785-795

.\xelsen. K.B. and Palmgren, M.G. (1998) Evolution of substrate specificities in the P- t>pe ATPase superfamily. J A/o/ Evol, 46, 84-101.

Barrack, E.R. (1987) Steroid hormone receptor localization in the nuclear matrix: interaction with acceptor sites. J Steroid Biochem., 27, 115-121.

Belmont, .A.S., Zhai, Y and Thilenius, A. (1993) Lamin B distribution and association with peripheral chromatin revealed by optical sectioning and electron microscopy tomography. JCW/5/o/, 123, 1671-1685.

Berezney, R. and Coffey, D.S. (1974) Identification of a nuclear protein matrix. Biochem Biophys Res Commun, 60, 1410-1417.

Berrios, M., Osheroff N. and Fisher, P.A. (1985) In situ localization of DNA topoisomerase II, a major polypeptide component ofthe Drosophila nuclear matrix fraction. Proc Nad Acad Sci USA, 82, 4142-4146.

Bidwell, J.P., Van Wijnen, A.J., Fey, E.G., Dworetzky, S., Penman, S., Stein, J.L., Lian, J.B. and Stein, G.S. (1993) Osteocalcin gene promoter-binding factors are tissue- specific nuclear matrix components. Proc Nad Acad Sci USA, 90, 3162-3166.

Bione, S., Maestrini, E., Rivella, S., Mancini, M., Regis, S., Romeo, G. and Toniolo, D. (1994) Identification of a novel X-linked gene responsible for Emery-Dreifiass muscular dystrophy. Nat Genet, 8, 323-327.

Borden, K.L. (2000) RING domains: master builders of molecular scaffolds? J Mo/ Biol, 295. 1103-1112.

Borden, K.L. and Freemont, P.S. (1996) The RING finger domain: a recent example of a sequence-structure family. Curr Opin Struct Biol, 6, 395-401.

Boyer, L.A. and Peterson, CL. (2000) Actin-related proteins (Arps): conformational switches for chromatin- remodeling machines? Bioessays, 22, 666-672.

11 <^'arter, K.C, Bowman, D., Carrington, W., Fogarty, K., McNeil, J.A., Fay, F.S. and Lawrence, J.B. (1993) A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus [see comments] Science 259 1330- 1335

C\)ok, P.R. (1999) The organization of replication and transcription. Science 284 1790- 1795.

Dechat, T., Gotzmann, J.. Stockinger, A., Harris, C.A., Talle, M.A., Siekierka, J.J. and Foisner, R. (1998) Detergent-salt resistance of LAP2alpha in interphase nuclei and phosphorylation-dependent association with chromosomes early in nuclear assembl\ implies functions in nuclear structure dynamics. Embo J, 17, 4887- 4902.

Ding, H.. Descheemaeker, K., Marynen, P., Nelles, L., Carvalho, T., Carmo-Fonseca, M., Collen, D. and Belayew, A. (1996) Characterization of a helicase-like transcription factor involved in the expression ofthe human plasminogen activator inhibitor-1 gene. DNA Cell Biol, 15, 429-442.

Doucas, V. (2000) The promyelocytic (PML) nuclear compartment and transcription control [In Process Citation]. Biochem Pharmacol, 60, 1197-1201.

Dyck. J.A.. Maul, G.G., Miller, W.H., Jr., Chen, J.D., Kakizuka, A. and Evans, R.M. (1994) A novel macromolecular structure is a target ofthe promyelocyte- retinoic acid receptor oncoprotein. Cell, 76, 333-343.

Fisher, D.Z.. Chaudhary, N. and Blobel, G. (1986) cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins. Proc Nad Acad Sci USA, 83, 6450-6454.

Freemont, P.S. (1993) The RING finger. A novel protein sequence motif related to the zinc finger. Ann N YAcad Sci, 684, 174-192.

Freemont, P.S.. Hanson, I.M. and Trowsdale. J. (1991) A novel cysteine-rich sequence motif [letter]. Cell, 64, 483-484.

Furukawa, K.. Pante, N., Aebi, U. and Gerace, L. (1995) Cloning of a cDNA for lamina- associated polypepfide 2 (LAP2) and identification of regions that specify targeting to the nuclear envelope. Embo J, 14, 1626-1636.

Gall, J.G., Tsvetkov, A., Wu, Z. and Murphy, C (1995) Is the sphere organelle/coiled body a universal nuclear component? Dev Genet, 16, 25-35.

112 Gla.ss, C.A., Glass, J.R., Taniura, II., Hasel, K.W., Blevitt, J.M. and Gerace, F. (1993) The alpha-helical rod domain of human lamins A and C contains a chromatin binding site. Embo ,/, 12, 4413-4424.

Gotzmann, J. and Foisner, R. (1900) Lamins and lamin-binding proteins in functional chromatin organization. Cril Rev Eukaryol Gene Expr, 9, 257-265.

Gruenbaum, Y.. Wilson, K.L., llarel. A., Goldberg, M. and Cohen, M. (2000) Review: nuclear lamins-structural proteins with fundamental functions. J Struct Biol, 129, -J2.I.

Hayward-Lester, .\.. Hewetson, A., Beale, E.G., Oefner, P.J., Doris, P.A. and Chilton, B.S. (1996) Cloning, characterization, and steroid-dependent posttranscriptional processing of RUSH-1 alpha and beta, two uteroglobin promoter-binding proteins. Mol Endocrinol, 10, 1335-1349.

Hendzel, M.J.. Boisvert, F. and Bazett-Jones, D.P (1999) Direct visualization of a protein nuclear architecture. Mol Biol Cell, 10, 2051-2062.

Hendzel, M.J.. Delcuve, G.P and Davie, J.R. (1991) Histone deacetylase is a component ofthe internal nuclear matrix. J Biol Chem, 266, 21936-21942.

Iborra, F.J.. Jackson, D.A. and Cook, P.R. (1998) The path of transcripts from extra- nucleolar synthetic sites to nuclear pores: transcripts in transit are concentrated in discrete structures containing SR proteins. J Cell Sci, 111, 2269-2282.

Jackson, D.A., Hassan, A.B., Errington, R.J. and Cook, P.R. (1993) Visualization of focal sites of transcription within human nuclei. Embo J, 12, 1059-1065.

Jackson. D.A., Iborra, F.J., Manders, E.M. and Cook, P.R. (1998) Numbers and organization of RNA polymerases, nascent transcripts, and transcription units in HeLa nuclei [published erratum appears in Mol Biol Cell 1998 Sep;9(9):2698]. Mol Biol Cell 9, 1523-1536.

Kleis-SanFrancisco, S., Hewetson, A. and Chilton, B.S. (1993) Prolactin augments progesterone-dependent uteroglobin gene expression by modulating promoter- binding proteins. Mol Endocrinol, 7, 214-223.

Kyte, J. and Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol, 157, 105-132.

Landers, J.P. and Spelsberg, T.C. (1992) New concepts in steroid hormone action: transcription factors, proto- oncogenes, and the cascade model for steroid regulation of gene expression. Crit Rev Eukaryot Gene Expr, 2, 19-63.

113 Ma, IF, Siegel, A.J. and Berezney, R. (1999) Association of chromosome territories with the nuclear matrix. Disruption of human chromosome territories correlates with the release of a subset of nuclear matrix proteins. J Cell Biol, 146, 531-542.

Mancini, M..\., Shan, B., Nickerson, J.A., Penman, S. and Lee, W.H. (1994) The retinoblastoma gene product is a cell cycle-dependent, nuclear matrix-associated protein. Proc \atl.4cadSci USA, 91, 418-422.

Martin, L., Crimaudo, C and Gerace. F. (1995) cDNA cloning and characterization of lamina-associated polypeptide IC (LAPIC), an integral protein ofthe inner nuclear membrane. J Biol ('hem, 270, 8822-8828.

Misteli, T., Caceres, J.F. and Spector, D.L. (1997) The dynamics of a pre-mRNA splicing factor in li\ ing cells. Xaliirc, 387, 523-527.

Mittnacht, S. and Weinberg, R.A. (1991) Gl/S phosphorylation ofthe retinoblastoma protein is associated with an altered affinity for the nuclear compartment. Cell, 65,381-393.

Moller, J.v.. Juul, B. and le Maire, M. (1996) Structural organization, ion transport, and energy transduction of P- type ATPases. Biochim Biophys Acta, 1286, 1-51.

Nakayasu, H. and Berezney, R. (1989) Mapping replicational sites in the eucaryotic cell nucleus. J Ce// Biol, 108, 1-11.

Nakayasu, H. and Ueda, K. (1983) Association of actin with the nuclear matrix from bovine lymphocytes. Exp Cell Res, 143. 55-62.

Nakayasu, H. and Ueda, K. (1985) Ultrastructural localization of actin in nuclear matrices from mouse leukemia L5178Y cells. Cell Struct Funct, 10, 305-309.

Nickerson, J.A., Blencowe, B.J. and Penman, S. (1995) The architectural organization of nuclear metaboUsm. Int Rev Cytol 67-123.

Nickerson, J.A., Krockmalnic, G., Wan, K.M. and Penman, S. (1997) The nuclear matrix revealed by eluting chromatin from a cross-linked nucleus. Proc Nad Acad Sci U 5^,94,4446-4450.

Papoulas O.. Beek, S.J., Moseley, S.L., McCallum, CM., Sarte, M.. Sheam, A. and Tamkun, J.W (1998) The Drosophila trithorax group proteins BRM, ASHl and ASH2 are subunits of distinct protein complexes. Development, 125, 3955-3966.

Paulin-Levasseur, M., Blake, D.L., Julien, M. and Rouleau, L. (1996) The MAN antigens are non-lamin constituents ofthe nuclear lamina in vertebrate cells. Chromosome. 104, 367-379.

114 Peterson, C.L., Zhao, Y. and Chait, B. 1 (1998) Subunits ofthe yeast SWI/SNF complex are members ofthe actin-related protein (ARP) family. J Biol ('hem. 273, 23641- 23044.

Pombo, .\. and Cook, P.R. (U)^)0) Fhe localization of sites containing nascent RNA and splicing factors. Exp Cell Res, 229, 201-203.

Pombo. A.. Cuello, P., Schul, W., Voon, J.B., Roeder, R.G., Cook, P.R. and Murphy, S. (1998) Regional and temporal specialization in the nucleus: a transcriptionally- actixe nuclear domain rich in PTF, Octl and PIKA antigens associates with specific chromosomes early in the cell cycle. Embo J, 17, 1768-1778.

Robinson, C..\.. Hayward-Lester. A., Hewetson, A., Oefner, P.J., Doris, P.A. and Chilton. B.S. (1997) Quantification of alternatively spliced RUSH mRNA isoforms b> QRT-PCR and IP-RP-HPLC analysis: a new approach to measuring regulated splicing efficiency. Gene, 198, 1-4.

Sasse\ ille, .A.M. and Langelier, Y. (1998) In vitro interaction ofthe carboxy-terminal domain of lamin A with actin. FEBS Lett, 425, 485-489.

Saurin, A.J., Borden, K.L., Boddy, M.N. and Freemont, P.S. (1996) Does this have a familiar RING? Trends Biochem Sci, 21, 208-214.

Schuchard, M., Rejman, J.J., McCormick, D.J., Gosse, B., Ruesink, T. and Spelsberg, T.C. (1991) Characterization of a purified chromatin acceptor protein (receptor binding factor 1) for the avian oviduct progesterone receptor. Biochemistry, 30, 4535-4542.

Stief A., Winter, D.M., Strafling, W.H. and Sippel, A.E. (1989) A nuclear DNA attachment element mediates elevated and position- independent gene activity. Nature, 341, 343-345.

Smurman, N., Heins, S. and Aebi, U. (1998) Nuclear lamins: their structure, assembly, and interactions. J 5^rwc/ Biol, 122, 42-66.

Tang, X.. Halleck, M.S., Schlegel, R.A. and Williamson, P. (1996) A subfamily of P-type ATPases with aminophospholipid transporting activity [published erratum appears in Science 1996 Dec 6;274(5293):1597]. Science, 272, 1495-1497.

Taniura, H., Glass, C and Gerace, L. (1995) A chromatin binding site in the tail domain of nuclear lamins that interacts with core histones. J Ce// Biol, 131, 33-44.

115 van Wijnen, A.J., Bidwell, J.P., Fey, E.G., Penman, S., Lian, J.B., Stein, J.L. and Stein, Ci.S. (1003) Nuclear matrix association of multiple sequence-specific DNA binding activities related to SP-1, ATF, CCAAT, C/EBP, OCT-1, and AP-1. Biochcmislrv, 32, 8397-8402.

\incent, M., LauriauU. P., Dubois, M.F., Lavoic, S., Bensaude, O. and Cbabot, B. (1996) The nuclear matrix protein p255 is a highly phosphorylated form of RNA polymerase 11 largest subunit which associates with spliceosomes. Nucleic Acids Res, 24. 4649-4652.

Wei. X., Somanathan, S., Samarabandu, J. and Berezney, R. (1999) Three-dimensional visualization of transcription sites and their association with splicing factor-rich nuclear speckles. J CV// Biol, 146, 543-558.

\\ orman. H.J., Yuan, J., Blobel, G. and Georgatos, S.D. (1988) A lamin B receptor in the nuclear envelope. Proc Nad Acad Sci USA, 85, 8531-8534.

Zbarskii, LB. and Debov, S.S. (1948) On the proteins ofthe cell nucleus. Dokl. Akad. Nauk SSSR, 63, 795-798.

Zhang, Q., Ekhterae, D. and Kim, K.H. (1997) Molecular cloning and characterization of Pl 13, a mouse SNF2/SWI2- related transcription factor. Gene, 202, 31-37.

Zhao, K.. Harel, A., Stuurman, N., Guedalia, D. and Gruenbaum, Y. (1996) Binding of matrix attachment regions to nuclear lamin is mediated by the rod domain and depends on the lamin polymerization state. FEBS Lett, 380, 161-164.

Zhao, K., Wang, W., Rando, O.J., Xue, Y., Swiderek, K., Kuo, A. and Crabtree, G.R. (1998) Rapid and phosphoinositol-dependent binding ofthe SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling. CeU, 95, 625-636.

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