UC San Diego Electronic Theses and Dissertations

UC San Diego UC San Diego Electronic Theses and Dissertations

Title Mechanisms of U6 small nuclear RNA gene expression in Drosophila melanogaster

Permalink https://escholarship.org/uc/item/89r1v4c0

Author Verma, Neha

Publication Date 2017

Peer reviewed|Thesis/dissertation

eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO SAN DIEGO STATE UNIVERSITY

Mechanisms of U6 small nuclear RNA gene expression in Drosophila melanogaster

A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in

Biology

Neha Verma

Committee in Charge:

University of California, San Diego

Professor Randolph Y. Hampton Professor Katherine A. Jones

San Diego State University

Professor William E. Stumph, Chair Professor Sanford I. Bernstein Professor Terrence Frey

2017

The Dissertation of Neha Verma is approved, and it is acceptable in quality and form for publication on microfilm and electronically:

______

Chair

University of California, San Diego San Diego State University

2017

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DEDICATION This dissertation is dedicated to my parents, my husband, my in-laws, the rest of my family and my loving friends for their incredible support, continuous encouragement, and endless love.

TABLE OF CONTENTS

Signature Page ...... iii Dedication Page ...... iv Table of Contents ...... v List of Figures ...... vii Acknowledgements ...... ix Vita ...... xi Abstract of the Dissertation ...... xiv

General Introduction ...... 1 A: References ...... 13

Chapter 1 Differential utilization of TATA box-binding protein (TBP) and TBP- related factor 1 (TRF1) at different classes of RNA polymerase III promoters ... 18 A: Abstract ...... 19 B: Introduction ...... 19 C: Materials and Methods ...... 20 D: Results ...... 21 E: Discussion ...... 24 F: References ...... 25

Chapter 2 SNAPc interacts with Bdp1 to establish a stable protein-DNA complex with TBP on a U6 snRNA gene promoter ...... 27 A: Introduction ...... 28 B: Materials and Methods ...... 31 C: Results ...... 34 D: Discussion ...... 45 E: References ...... 48

Chapter 3 Mapping a region of SNAPc responsible for recruitment of Bdp1 to the . Drosophila melanogaster U6 snRNA gene promoter ...... 53 A: Introduction ...... 54 B: Materials and Methods ...... 57 C: Results and Discussion ...... 59 D: Refernces ...... 61

Concluding Remarks ...... 63 A: References ...... 70

Appendix ...... 72 A: Detailed protocol for chromatin immunoprecipitation assay (ChIP) ... 73 B: Detailed protocol for in vitro transcription assay ...... 85 C: Detailed protocol for immune-depletion assay ...... 100 D: Detailed protocol for nickel-chelate chromatography ...... 106 E: Detailed protocol for electrophoretic mobility shift assay (EMSA) .... 115

LIST OF FIGURES

General Introduction

Figure 1: Schematic representation of cis-acting elements in the 5’- flanking DNA of a variety of snRNA genes ...... 2 Figure 2: Comparison of different classes of D. melanogaster snRNA gene promoters ...... 4 Figure 3: Working model for RNA polymerase specificity at U1 (A) and U6 (B) snRNA promoters ...... 7

Chapter 1

Figure 1: ChIPs indicating that D. melanogaster U6 snRNA gene promoters are occupied by TBP in vivo ...... 21 Figure 2: U6 snRNA transcription in vitro utilizes TBP ...... 23 Figure 3: Manipulation of TBP and TRF1 levels in cells by overexpression (A) and by RNAi knockdown (B) ...... 24

Chapter 2

Figure 1: Assembly of DmSNAPc with individual subunits of TFIIIB on the U6 snRNA gene promoter ...... 35 Figure 2: A region of Bdp1 C-terminal of the SANT domain is required for its recruitment by DmSNAPc ...... 37 Figure 3: A Pol III-specific PSEA is necessary and sufficient for Bdp1 recruitment by DmSNAPc ...... 39 Figure 4: The DmSNAPc-Bdp1 complex efficiently recruits TBP to the U6 promoter ...... 42 Figure 5: A TATA box is required for recruitment of TBP to the U6 promoter by DmSNAPc-Bdp1 ...... 43

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Figure 6: Mapping a region of Bdp1 that participates in the recruitment of TBP by the DmSNAPc-Bdp1 complex on U6 promoter DNA ...... 44 Figure 7: Bdp1 is required, together with DmSNAPc bound to a Pol III-specific PSEA and TBP bound to a TATA box, to form a stable protein-DNA complex on the bipartite Drosophila U6 snRNA gene promoter ...... 46

Chapter 3

Figure 1: Schematic representation of various DmSNAP43 constructs ...... 56 Figure 2: Mapping a region of DmSNAPc involved in interaction with Bdp1 on U6 promoter DNA ...... 58

Concluding Remarks

Figure 1: Schematic representation of transcription requirements at different classes of RNA polymerase III promoters ...... 66

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ACKNOWLEDGEMENTS

First of all, I would like to thank my thesis advisor Dr. Stumph for his mentorship, guidance, motivation and immense support for the years I have been in his lab. I learnt a lot and grew as a scientist under his leadership and could not have imagined a better advisor for my PhD.

Besides my advisor, I would also like to thank Dr. Bernstein, Dr. Frey, Dr.

Hampton and Dr. Jones for serving on my dissertation committee and giving me invaluable suggestions on my projects to make this thesis possible.

I am grateful to all my past and current lab mates at San Diego State University for their help and encouragement that facilitated the accomplishment of my projects and made this experience so much fun. I thank Ko-Hsuan Hung and Jin Joo Kang for their contributions to the publication that constitutes Chapter 1 of this thesis. I am also grateful to Ann Marie Hurlburt, Yoon Soon Kang, Phuc Phan, and Angela Wolfe for their assistance in preparation of Bdp1 truncation constructs and performing nickel- chelate chromatography and EMSAs for Chapter 2 of this thesis.

I would like to thank my parents for giving birth to me in the first place and also for supporting me throughout this journey. I am also deeply grateful to my husband,

Sidhanshu Mendiratta, for his endless support, late night visits to lab and constant encouragement to overcome each roadblock that came in my way throughout this process.

Chapter one, in full, is a reprint of the material as it appears in The Journal of

Biological Chemistry, 2013. Differential utilization of TATA box-binding protein

(TBP) and TBP-related factor 1 (TRF1) at different classes of RNA polymerase III

promoters. Verma, N., Hung, K. H., Kang, J. J., Barakat, N. H., Stumph, W. E. The dissertation author was the primary researcher and author of this paper.

Chapter two is written as a manuscript to be submitted for publication immediately following approval of this thesis. SNAPc interacts with Bdp1 to establish a stable protein-DNA complex with TBP on a U6 snRNA gene promoter. The dissertation author was the primary author of this manuscript.

The material in chapter three, is not intended to be submitted for publication at the moment. The dissertation author was the primary researcher and author of this chapter.

The material described in this written dissertation is based upon work supported by the National Science Foundation under Grant Numbers 1157549 and 1616487. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author and do not necessarily reflect the views of the National Science

Foundation.

VITA

2004-2008 B.Tech, Biotechnology with Honors CET-IILM-AHL Greater Noida, UP, India 2006 Research Summer Intern Department of Quality Control, Dabur India Ltd Ghaziabad, UP, India 2007 Research Summer Intern Department of Biology, Clonegen Biotechnology Pvt Ltd Noida, UP, India 2008 Research Summer Intern Department of Bioinformatics, Bioinformatics Institue of India Noida, UP, India 2009-2012 M.S. Program Cell and Molecular Biology Department of Biology, San Diego State University San Diego, California 2009-2017 Teaching Associate Department of Biology, San Diego State University San Diego, California 2012-2015 Pre-doctoral Candidate Department of Biology (Cell and Molecular Biology) University of California, San Diego and San Diego State University San Diego, California 2015-2017 Doctoral Candidate

Department of Biology (Cell and Molecular Biology) University of California, San Diego and San Diego State University San Diego, California 2017 Ph.D. in Biology University of California, San Diego and San Diego State University San Diego, California

PUBLICATIONS

Verma N, Hurlburt AM, Kang, YS, Phan P and Stumph WE (In preparation) SNAPc interacts with Bdp1 to establish a stable protein-DNA complex with TBP on a U6 snRNA gene promoter.

Verma N, Hung KH, Kang, JJ, Barakat, NH and Stumph WE (2013) Differential utilization of TATA box-binding protein (TBP) and TBP-related factor 1 (TRF1) at different classes of RNA polymerase III promoters. J. Biol. Chem. 288.38, 27564- 27570

CONFERENCE ABSTRACTS

Hurlburt AM, Verma N, and Stumph WE (2017). "Bdp1 interacts with SNAPc on a U6 snRNA gene promoter and functions as an intermediate to form a stable protein complex with TBP" Cold Spring Harbor Laboratory Meeting on “Mechanisms of Eukaryotic Transcription” at Cold Spring Harbor, NY

Verma N, Hurlburt AM, Kang JJ, and Stumph WE (2017). "Assembly of SNAPc and TFIIIB on a Drosophila U6 snRNA gene promoter" CSU Annual Biotechnology Symposium, Santa Clara, CA.

Verma N, Hung KH, Kang JJ, and Stumph WE (2016). "Differential Utilization of TATA Box Binding Protein (TBP) and TBP-related Factor 1 (TRF1) at Different Classes of RNA Polymerase III Promoters." CSU Annual Biotechnology Symposium, Orange County, CA.

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Verma N, Hung KH, Kang JJ, and Stumph WE (2014). "Differential Utilization of TATA Box Binding Protein (TBP) and TBP-related Factor 1 (TRF1) at Different Classes of RNA Polymerase III Promoters." International Conference on Transcription by RNA Polymerase I, III, IV, and V: OddPols 2014, Ann Arbor, MI.

Verma N, Hung KH, Kang JJ, and Stumph WE (2014). "Differential Utilization of TATA Box Binding Protein (TBP) and TBP-related Factor 1 (TRF1) at Different Classes of RNA Polymerase III Promoters." 55th Annual Drosophila Research Conference. San Diego, CA.

Hung KH, Verma N, Kang JJ, and Stumph WE (2013). "Differential Utilization of TATA Box-Binding Protein (TBP) and TBP-related Factor 1 (TRF1) at Different Classes of RNA Polymerase III Promoters." Cold Spring Harbor Laboratory Meeting on “Mechanisms of Eukaryotic Transcription” at Cold Spring Harbor, NY.

FIELDS OF STUDY

Major Field: Cell and Molecular Biology

Studies in Transcriptional Regulation of Eukaryotic Gene Expression Professor William E. Stumph

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ABSTRACT OF THE DISSERTATION

Mechanisms of U6 small nuclear RNA gene expression in Drosophila melanogaster by Neha Verma

Doctor of Philosophy in Biology University of California, San Diego, 2017 San Diego State University, 2017

Professor William E. Stumph, Chair

The goal of this study involves determining the requirements and mechanisms of

RNA polymerase-specific transcription complex assembly on U6 small nuclear RNA

(snRNA) gene promoters in Drosophila melanogaster. In higher eukaryotes, RNA polymerase III (Pol III) promoters at U6 snRNA genes consist of a TATA box, recognized by TFIIIB and a proximal sequence element, PSE, recognized by the small nuclear RNA activating protein complex (SNAPc). In Drosophila melanogaster,

DmSNAPc consists of three subunits DmSNAP190, DmSNAP50, and DmSNAP43; likewise TFIIIB also consists of three subunits, most commonly TBP, Brf1 and Bdp1.

Tjian’s lab [Takada et al., 2000] concluded that TRF1 (TBP-related factor 1), instead of

TBP, was utilized at all Pol III promoters (including U6 transcription) in D. melanogaster. However, preliminary work in our lab on U6 caused us to question this widely accepted notion.

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Chapter 1 elucidates the transcriptional requirements of Drosophila melanogaster U6 snRNA genes with reference to solve the discrepancy between the two general transcription factors (GTFs), TBP and TRF1. Our results revealed that although

TRF1 mediates Pol III-dependent transcription of the tRNA genes, transcription of the

U6 snRNA genes in contrast utilizes TBP [Verma et al., 2013] indicating the differential utilization of TBP and TRF1 at different classes of Pol III promoters.

Chapter 2 describes a study that provided a mechanistic explanation of the Pol

III specificity of U6 snRNA gene promoters by investigating the existence of interactions between DmSNAPc and TFIIIB on the U6 promoter. Our results showed that DmSNAPc, when bound to a U6 promoter (but not to a U1 promoter), attains the ability to recruit Bdp1 and subsequently TBP for Pol III transcription.

Chapter 3 studies various truncation and alanine-scanning mutations of

DmSNAPc in an attempt to identify regions involved in the novel interaction between

DmSNAPc and Bdp1 on the U6 promoter as reported in Chapter 2. Despite utilizing a variety of alanine-scanning constructs of DmSNAP43 (the subunit of DmSNAPc that cross-links to the DNA furthest downstream of the U6 PSEA and hence closest to

TFIIIB binding site on the U6 promoter), we were unable to detect a region responsible for Bdp1 recruitment.

GENERAL INTRODUCTION

1 2

The snRNA genes – a unique class of transcription units.

The snRNAs known as U1, U2, U4, U5, and U6 comprise a highly abundant class of metabolically stable non-polyadenylated RNA molecules that are required for pre-mRNA splicing [Guthrie, 1991; Sharp, 1994]. These snRNAs are synthesized by

Pol II, with the exception of U6, which is synthesized by Pol III [Parry et al., 1989].

The snRNA genes in all organisms studied (with the likely exception of the yeast

Saccharomyces cerevisiae) have promoter structures and cis-acting elements that functionally distinguish them from classical Pol II or Pol III transcription units.

Figure 1: Schematic representation of cis-acting elements in the 5’-flanking DNA of a variety of snRNA genes. All the snRNA genes shown contain an essential element upstream of position -40. Many snRNA genes also contain a regulatory element near position -30 or -25, which probably represents a site of interaction with TBP

Fig. 1 summarizes work on a variety of higher eukaryotes and shows regulatory elements identified in the basal promoter regions of snRNA genes transcribed by either Pol II or Pol III. A promoter element (designated PSE, PSEA, or

USE in Fig. 1) is located approximately 40 to 75 bp upstream of the start site and is

3 essential for the initiation of snRNA gene transcription [Das et al., 1987; Parry et al.,

1989; Zamrod et al., 1993].

Besides the PSE, many snRNA genes contain a second promoter element located approximately 20-30 bp upstream of the transcription start site. An exception is that vertebrate Pol II-transcribed snRNA genes lack any well-conserved element in this region (Fig. 1). In contrast, vertebrate U6 genes, transcribed by Pol III, contain a conserved TATA box. Mutation of the U6 TATA box to an unrelated sequence changed the promoter specificity from Pol III to Pol II [Lobo and Hernandez, 1989;

Mattaj et al., 1988]. Conversely, the introduction of a TATA sequence into the U1 or

U2 promoters altered their specificity to Pol III [Lobo and Hernandez, 1989; Mattaj et al., 1988]. These findings led to the idea that the TATA box acts as a dominant element in determining the Pol III specificity of vertebrate U6 promoters [Hernandez,

2001].

In plants, both classes of snRNA genes contain a TATA box. In this case, polymerase specificity is determined by a 10 bp difference in spacing between the

TATA box and the USE (32-36 bp spacing for Pol II versus 23-26 bp for Pol III, Fig.

1) [Waibel and Filipowicz, 1990]. In vertebrates and plants (as well as sea urchins), the PSE (or USE) was found to be functionally interchangeable among the U1, U2, and U6 genes [Lobo et al., 1989; Waibel and Filipowicz, 1990]. The conclusion from those studies was that the PSE or USE itself does not contribute directly to RNA polymerase specificity in those organisms. There is little or no mechanistic data about how RNA polymerase specificity is determined by the presence vs. absence of a

TATA box in vertebrate snRNA gene promoters, or by the difference in spacing between the USE and TATA box in plants.

In the fruit fly, the promoter elements of the various snRNA genes are more conserved with regard to both sequence and location than generally observed in other organisms (Fig. 2) [Jensen et al., 1998; Das et al., 1987]. All known Drosophila snRNA genes contain a 21 bp PSEA that is well conserved in sequence [Jensen et al.,

1998; Das et al., 1987]. All Drosophila snRNA genes transcribed by Pol II also contain a well-conserved 8 bp PSEB that contributes to efficient initiation of transcription [Zamrod et al., 1993]. A separation is strictly conserved of 8 bp between the PSEA and PSEB in Pol II-transcribed snRNA genes and 12 bp between the PSEA and TATA box of U6 gene promoters [Jensen et al., 1998; Das et al., 1987].

Figure 2: Comparison of different classes of D. melanogaster snRNA gene promoters. A, Conserved structure of Drosophila snRNA gene promoters transcribed by Pol II and Pol III. B, The wild type U1 and U6 PSEA sequences that we use in our studies differ at only 5 of 21 nucleotide positions. The PSEB and TATA elements differ at 5 of 8 positions. The sequences shown are from the U1:95Ca gene and the U6:96Ab gene.

The PSEA – a dominant element for determining the RNA

polymerase specificity of Drosophila snRNA gene promoters.

In vitro transcription using mix-and match templates that combined all possible combinations of U1 or U6 PSEA, 8 or 12 bp spacing, and PSEB or TATA box has been reported earlier from our lab [Jensen et al., 1998]. The U1 and U6 PSEAs used in our experiments differed at only the 5 nucleotide positions shown in Fig. 2. Constructs that contained the U1 PSEA were transcribed by Pol II, and those that contained the

U6 PSEA were transcribed by Pol III. The PSEB and TATA elements, as well as the 8 vs. 12 bp spacing, affected transcription efficiency but did not directly affect the choice of RNA polymerase in vitro [Jensen et al., 1998].

Analogous in vivo experiments were carried out with reporter constructs that contained U1 and U6 promoters with “swapped” PSEAs [McNamara-Schroeder et al.,

2001]. Substitution of the U6 PSEA into the U1 promoter, or substitution of the U1

PSEA into the U6 promoter suppressed transcription in vivo. The results clearly indicated that the U1 PSEA cannot function for Pol III transcription and the U6 PSEA cannot function for Pol II transcription, even though they differ at only 5 of 21 positions.

Trans-acting Factors that Assemble on the Basal Promoters of snRNA genes.

The Small Nuclear RNA Activating Protein Complex (SNAPc)

Human

The human PSE-binding protein or small nuclear RNA activating protein complex (SNAPc) binds to the PSE and is required for transcription of human U1 and

U2 genes by Pol II and of U6 genes by Pol III [Yoon et al., 1995; Sadowski et al.,

1993; Goomer et al., 1994]. Human SNAPc contains integral polypeptide subunits with apparent molecular weights of approximately 19, 43, 45, 50, and 190 kDa [Yoon and Roeder, 1996; Sadowski et al., 1996; Bai et al., 1996; Henry et al., 1996; Henry et al., 1998] and are referred to as SNAP19, SNAP43, SNAP45, SNAP50, and

SNAP190.

Drosophila

The Drosophila melanogaster SNAPc (DmSNAPc) is a hetero-trimeric transcription factor [Su et al., 1997] that is required for the synthesis of the U1, U2,

U4, U5, and U6 small nuclear RNAs (snRNAs) [Li et al., 2004; Barakat and Stumph,

2008; Hernandez et al., 2007]. DmSNAPc binds sequence-specifically to the conserved ~21 base pair (bp) promoter element termed the PSEA (Figs. 1 and 2) and can activate transcription of the Drosophila U1 and U6 snRNA genes in vitro

[Hernandez et al., 2007]. Our lab also showed that DmSNAPc contains three distinct polypeptides (DmSNAP43, DmSNAP50, and DmSNAP190) each of which contacts the DNA [Wang and Stumph, 1998]. Moreover, the face of the DNA helix that each of the subunits interacts with (relative to the PSEA sequence and to each other) was determined [Wang and Stumph, 1998]. It was found that DmSNAP190 contacts the entire length of the PSEA while DmSNAP43 and DmSNAP50 interact only with the

7 differences are differentially interpreted by the basal transcription machinery to confer promoter specificity for either Pol II or Pol III (Fig. 3) [Jensen et al., 1998;

McNamara-Schroeder et al., 2001; Lai et al., 2005; Barakat and Stumph, 2008].

Pol II GTFs

Pol III GTFs

Figure 3: Working model for RNA polymerase specificity at U1 (A) and U6 (B) snRNA promoters. Each subunit of DmSNAPc is drawn schematically to indicate the region of DNA with which it interacts based upon protein-DNA photocrosslinking data. We propose that the different conformations of DmSNAPc recruit different sets of general transcription factors (GTFs).

The General Transcription Factors (GTFs)

RNA Polymerase II Transcription

All the Pol II general transcription factors (TFIIA, TFIIB, TBP, TFIIE, and

TFIIF), with the possible exception of TFIIH [Kuhlman et al., 1999; Pavelitz et al.,

2008;], play a role in human U1 transcription. Presumably, they are also required for transcription of U1 snRNA in Drosophila melanogaster, although, with the exception of TBP, this has not been investigated either by our lab or others.

RNA Polymerase III Transcription

In higher eukaryotes, there are at least three distinct classes of promoters for

Pol III [Hernandez, 2001]. Type I and Type II Pol III promoters (exemplified by the

5S rRNA genes and tRNA genes, respectively) are internal to the genes and depend upon the DNA-binding complex TFIIIC for the assembly of a transcription preinitiation complex (PIC) [Geiduschek and Kassavetis, 2001; Schramm and Hernandez,

2002; Willis, 1993]. Type III Pol III promoters comprise sequences external to the gene located in the 5’-flanking DNA. This third type of Pol III promoter is exemplified by genes for U6 snRNA and certain other small stable RNAs [Geiduschek and Kassavetis, 2001; Schramm and Hernandez, 2002, Hernandez, 2001; Willis, 1993;

Jawdekar and Henry, 2008; Egloff et al., 2008].

Human U6 transcription requires TBP, Bdp1, and a unique form of a TFIIB- related factor known as Brf2 [Cabart and Murphy, 2001]. Brf2 is a paralog of Brf1, the factor that functions in human tRNA and 5s RNA transcription [Parry et al., 1989;

Mattaj et al., 1988; Wang and Roeder, 1995]. Together with TBP, Bdp1 and Brf1/Brf2 compose the factor known as TFIIIB.

Drosophila melanogaster TFIIIB (TBP/TRF1, Bdp1, Brf1)

TBP/TRF1

TBP used to be thought of as the ‘universal’ transcription factor [Crowley et al., 1993], required by all three RNA polymerases but the universality of TBP was questioned when TBP-related factors were discovered. The first of these, TBP-related

9 factor 1 (TRF1), was identified in Drosophila, and so far is unique to insects

[Geiduschek and Kassavetis, 2001]. TRF1 is highly related to the conserved core domain of TBP. Staining of polytene chromosomes showed that TRF1 and TBP localize to different loci and that TRF1 localizes to Pol III-transcribed loci.

Biochemical experiments showed that depletion of TRF1, but not TBP, in Drosophila embryo nuclear-extracts led to inhibition of Pol III transcription [Takada et al., 2000].

Furthermore, Isogai et al. [Isogai et al., 2007] suggested that unlike most other eukaryotic organisms that rely on TBP for Pol III transcription, in Drosophila (and probably in other insects) TRF1 appears responsible for the initiation of Pol III transcription.

Brf1 and Bdp1

Brf1 is a TFIIB-related factor that is specific for transcription by Pol III. It probably plays a role in Pol III transcription similar to the role of TFIIB in Pol II transcription. Interestingly, mammals have two forms of Brf (Brf1 and Brf2) encoded by different genes, and Brf2 is an snRNA-specific form of Brf that is required for transcription at Class III Pol III promoters [see references Geiduschek and Kassvetis,

2001; Schramm and Hernandez, 2002; for reviews]. However, the D. melanogaster genome contains only a single gene that codes for a Brf protein, and this protein corresponds to Brf1. Although all TFIIB family members share the function of bridging promoter-bound factors and the RNA polymerase, TFIIB and yeast Brf1 accomplish this function differently. TFIIB assembles with the TBP/TATA box complex through its conserved core domain [Yamashita et al., 1993], while the C-

10 terminus as well as the core domain of yeast Brf1 is involved in this interaction

[Kassavetis et al., 1997]. Furthermore, zinc ribbon domain is crucial for the recruitment of Pol II by TFIIB whereas, the zinc ribbon is not necessary for Pol III recruitment by Brf1 but instead plays an essential role later in transcription initiation

[Yamashita et al., 1993; Kassavetis et al., 1997].

Bdp1 has been shown to have little, if any, affinity for promoter DNA in the absence of TBP and Brf1 [Kassavetis et al., 1991; Saida, 2008]. However, once TBP and Brf1 are bound to a TATA box, Bdp1 can associate to form a stable complex

[Colbert et al., 1998]. The binding of yeast Bdp1 to the TBP-Brf1-TATA box complex induces a bend in the DNA between the TATA box and transcription start site, which has been postulated to contribute to the yeast Bdp1-dependent stabilization of the

TFIIIB-DNA complex by helping impede sliding of the DNA out of the complex

[Grove et al., 1999]. Bdp1 also plays a post-Pol III recruitment role. It has been shown to be indispensable for transcription due to its essential role in promoter opening

[Kassavetis et al., 1992]. Currently, very little is known about the structure of Bdp1 and how it assembles into the TFIIIB complex.

Questions investigated by the work described in this dissertation

To examine the requirements and mechanisms of transcription initiation assembly on the U6 snRNA gene promoters, it was necessary to first identify which general transcription factor; TBP or TRF1 gets recruited by DmSNAPc on the U6 snRNA promoter DNA. Chapter 1 describes work toward resolving the discrepancy

11 found in the published literature and preliminary work from our lab in reference to the two general transcription factors in question. To determine the presence of TBP or

TRF1 at different Pol III promoters in vivo, chromatin immuno-precipitation (ChIP) assays were performed utilizing all three potentially active U6 snRNA genes present in the Drosophila melanogaster genome. As controls, three gene loci each of U1 snRNA and tRNA were also analyzed. Antibodies against TBP, TRF1 and

DmSNAP43 (as a control for snRNA genes) were employed in ChIPs to examine the

U6 promoter occupancy in vivo by either traditional PCR followed by gel electrophoresis or quantitative PCR (qPCR). Immunodepletions of these factors were carried out prior to in vitro transcription analysis followed by primer extension assays to examine the role of TBP v/s TRF1 in U6, U1, and tRNA transcription. Analogous experiments utilizing RNAi knockdown were also performed. Results from this first chapter have been published in the Journal of Biological Chemistry (2013) and the dissertation author is the co-first author of this research.

The second major part of my research is based upon the intriguing earlier finding in our lab that the precise nucleotide sequence of the PSEA also plays a significant role in dictating the RNA polymerase specificity at a particular snRNA gene promoter (Fig. 3) [Jensen et al., 1998; McNamara et al., 2001; Lai et al., 2005].

Work reported in Chapter 2 studies the mechanisms that lead to the Pol III specificity at the U6 promoter by investigating whether interactions between DmSNAPc and

TFIIIB could be identified on the U6 promoter. The interactions of DmSNAPc with individual subunits of TFIIIB (TBP, Brf1, or Bdp1) were analyzed by electrophoretic

12 mobility shift assays (EMSAs). Since we believe that DmSNAPc attains different conformations on a U1 v/s a U6 promoter, utilizing DNA probes with switched PSEAs in place of of wild type U6 or U1 probes assessed the role of different PSEAs in Bdp1 recruitment. Similar EMSA experiments were performed to examine if DmSNAPc-

Bdp1 complex might play a role in TBP recruitment to the U6 promoter DNA containing either wild type TATA sequence or a mutated version of the TATA box.

Furthermore, a series of N-terminal and C-terminal truncations of Bdp1 were made to identify the regions involved in interaction with DmSNAPc as well as TBP. In vitro complex formation between DmSNAPc and various truncated Bdp1 constructs in the absence or presence of TBP was also examined by EMSAs. Results from Chapter 2 are being prepared for submission for publication, and the dissertation author is the primary researcher of this study.

Chapter 3 describes work that involves mapping the regions of DmSNAPc that might be involved in the interaction with Bdp1. The lab had on hand a variety of alanine-scanning constructs and a truncation construct of DmSNAP43 that I utilized for this study. EMSAs were performed utilizing all these constructs to locate the region of DmSNAP43 that might be involved in recruitment of Bdp1 to the U6 promoter. Results of this study are presented in Chapter 3 and are not yet intended to be submitted for publication because we have not yet identified a region of DmSNAPc responsible for its interaction with Bdp1.

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17 proximal cis-acting elements with stringent spacing requirements. Mol Cell Biol 13, 5918-5927.

CHAPTER 1

Differential utilization of TATA box-binding protein (TBP) and TBP-related factor 1 (TRF1) at different classes of RNA polymerase III promoters

18 19

ACKNOWLEDGEMENTS: This Chapter, in full, is a reprint of the material as it appears in The Journal of

Biological Chemistry, 2013. Differential utilization of TATA box-binding protein

(TBP) and TBP-related factor 1 (TRF1) at different classes of RNA polymerase III promoters. Verma, N., Hung, K. H., Kang, J. J., Barakat, N. H., Stumph, W. E. The dissertation author was the primary researcher and author of this paper. Quantitative

ChIP assays, and RNA interference (RNAi) and cellular expression aasays were carried out by Ko-Hsuan Hung, whereas the histogram to measure relative expression of genes and the immunoblot to measure the relative molar amounts of TBP and TRF1 in the purified fractions were generated by Jin Joo kang. All other data in this paper were produced by this dissertation author.

CHAPTER 2

SNAPc interacts with Bdp1 to establish a stable protein-DNA complex with TBP on a U6 snRNA gene promoter

27 28

INTRODUCTION Although RNA polymerase III (Pol III) promoters were originally identified as

DNA sequences internal to the 5S rRNA and tRNA genes (that is, located downstream of the transcription start site), many genes transcribed by Pol III have promoters that are located entirely in the 5’-flanking DNA (Geiduschek and Tocchini-Valentini,

1988; Schramm and Hernandez, 2002; Willis, 1993). Pol III genes with external promoters include metazoan genes that code for U6 and U6 atac snRNAs, 7SK RNA, tRNAsel, H1 and MRP RNAs, as well as a number of other small non-coding RNAs

(Baer et al., 1990; Canella et al., 2010; Carbon and Krol, 1991; Hung and Stumph,

2011; Jawdekar and Henry, 2008; Kunkel et al., 1986; Murphy et al., 1987; Myslinski et al., 2001; Yuan and Reddy, 1991). These gene-external Pol III promoters are bipartite, consisting of both a TATA box and a proximal sequence element (PSE) centered about 30 base pairs (bp) and 55 bp upstream of the transcription start site respectively (Hung and Stumph, 2011; Jawdekar and Henry, 2008; Schramm and

Hernandez, 2002). The TATA box in this class of Pol III genes is a binding site for the three-subunit general transcription factor TFIIIB (Kang et al., 2016; Saxena et al.,

2005) that in mammals consists of TBP, Brf2, and Bdp1. Brf2 is paralogous to the more evolutionarily ancient Brf1 protein. Whereas Brf1 is used at mammalian Pol III- transcribed genes with internal promoters (e.g., tRNA and 5S RNA genes), Brf2 is instead used specifically at genes with upstream promoters (e.g., U6 and 7SK)

(Schramm et al., 2000; Teichmann et al., 2000; Willis, 2002).

The PSE on the other hand is the binding site for the multi-subunit small nuclear RNA activating protein complex (SNAPc) (Henry et al., 1995; Sadowski et

29 al., 1993), also known as PTF (Murphy et al., 1992; Yoon et al., 1995). Three subunits of SNAPc (SNAP43, SNAP50, and SNAP190) appear to be conserved throughout metazoan evolution (Hung and Stumph, 2011). Despite their role in Pol III transcription, the PSE and SNAPc are also essential for the transcription of snRNA genes transcribed by RNA polymerase II (Pol II) (e.g., U1 to U5 genes) (Egloff et al.,

2008; Henry et al., 1998; Hung and Stumph, 2011; Jawdekar and Henry, 2008; Parry et al., 1989).

Early studies in the human system revealed that SNAPc recruited TBP to the

U6 gene promoter through cooperative binding interactions (Mittal and Hernandez,

1997). It was also discovered that SNAPc interacted with and recruited Brf2 to U6 promoter DNA (Hinkley et al., 2003). By acting together, SNAPc and Brf2 incorporated limiting levels of TBP into a more stable SNAPc-Brf2-TBP-U6 promoter

DNA complex (Hinkley et al., 2003; Ma and Hernandez, 2002; Saxena et al., 2005).

However, to our knowledge, no direct interactions between Bdp1 and SNAPc were reported.

In fruit flies, no protein orthologous to mammalian Brf2 is encoded in the

Drosophila melanogaster genome; fruit flies therefore utilize Brf1 at both upstream and internal Pol III promoters (Isogai et al., 2007). Interestingly, however, a difference does exist in the basal transcription machinery utilized at fly genes with internal and external Pol III promoters: those with internal promoters utilize the TBP-related factor

TRF1, but those with upstream promoters such as U6 employ the canonical TBP

(Verma et al., 2013). In summary, the TFIIIB complex at U6 and U6-like promoters in flies consists of TBP, Brf1, and Bdp1.

A second line of studies by our lab has shown that the PSEs (more specifically referred to as PSEAs in fruit flies) of D. melanogaster U1 and U6 snRNA genes are not interchangeable. That is, a U1 PSEA will not support Pol III transcription of a U6 gene, and a U6 PSEA will not support Pol II transcription of a U1 gene, even though they are identical at 16 of 21 nucleotide positions and both are bound by DmSNAPc

(Barakat and Stumph, 2008; Hung and Stumph, 2011; Jensen et al., 1998; Lai et al.,

2005; McNamara-Schroeder et al., 2001). Furthermore, site-specific protein-DNA photo-cross-linking studies have indicated that D. melanogaster SNAPc (DmSNAPc) cross-links in distinctive manners to the PSEAs of U6 genes versus U1 genes (Hung and Stumph, 2011; Kang et al., 2014; Kim et al., 2010a, b; Lai et al., 2005; Li et al.,

2004; Wang and Stumph, 1998).

The above data have led to a model in flies in which the specific PSEA sequence, U1 or U6, acts as an allosteric effector of DmSNAPc conformation that leads to RNA polymerase specificity (Hung and Stumph, 2011; Kang et al., 2014; Kim et al., 2010a, b; Li et al., 2004; Wang and Stumph, 1998). Here we describe experiments that extend this model by providing a further mechanistic explanation for the Pol III specificity of U6 snRNA gene promoters. Our data reveal a newly- identified interaction between DmSNAPc and the Bdp1 subunit of TFIIIB that leads to stable complex formation together with TBP on U6 but not on U1 promoters.

MATERIALS AND METHODS

DmSNAPc Expression Constructs

The preparation of untagged constructs encoding wild type DmSNAP 43,

DmSNAP50, and DmSNAP190 as well as N-terminal His6-FLAG-tagged

DmSNAP43 or DmSNAP190 constructs under the control of the copper-inducible metallothionein promoter have been previously described (Hung et al., 2009).

Expression plasmids for each of the DmSNAPc subunits were used to co-transfect S2 cells as previously described (Hung et al., 2009). Subunit co-expression was induced with copper sulfate and confirmed by immunoblotting by using anti-FLAG M2 monoclonal antibodies (Sigma #A9469) or antibodies made against synthetic peptides corresponding to amino acid sequence at or near the C-terminus of the wild type proteins (Li et al., 2004).

TBP and Bdp1 Expression Constructs

TBP and Bdp1 constructs that encode the full-length C-terminal V5-His6- tagged TBP and C-terminal FLAG-Myc-His6-tagged Bdp1 respectively under the control of the copper-inducible metallothionein promoter have been previously described (Kang et al., 2016). Bdp1 truncation constructs were prepared by PCR, restriction digestion, and re-cloning as previously described for the DmSNAPc subunit truncation constructs (Hung et al., 2009). Expression plasmids were then used to generate stably transfected Drosophila S2 cell lines (Hung et al., 2009). Copper sulfate induced expression was confirmed by immunoblotting by using anti-V5 monoclonal antibodies (Life Technologies #R960-25) and anti-Myc monoclonal antibodies (Sigma

#M4439) respectively. Bacterially-expressed untagged recombinant Bdp1, used in some experiments, was expressed and purified by Genscript.

Protein Purification

Following copper sulfate induction, cells (sixteen 15 cm diameter plates per cell line grown to ~100% confluency) were lysed in CelLytic M lysis buffer (Sigma

#C2978) containing 1% protease inhibitor cocktail (Sigma # P8340). Lysates were then adjusted to a NaCl concentration of 0.5 M prior to incubating with ProBond resin

(Life Technologies #R80101) for 2 hrs for DmSNAPc and TBP proteins and 30 min for Bdp1 proteins to allow the capture of His6-tagged DmSNAPc, Bdp1, or TBP protein. The resins were then washed three times in 50 mM sodium phosphate buffer

(pH 8.0), 0.5 M NaCl, 20 mM imidazole and then once in HEMG-100 buffer (100 mM KCl, 25 mM HEPES K+ (pH 7.6), 12.5 mM MgCl2, 10 µM ZnCl2, 0.1 mM

EDTA (pH 8.0), 10% glycerol, and 0.5 mM phenylmethylsulfonyl fluoride) with 20 mM imidazole. The proteins/complexes were eluted from the resin with 750 mM imidazole in HEMG-100 buffer followed by addition of 3 mM dithiothreitol to each elution and then dialyzed against HEMG-100 buffer without imidazole but containing

1 to 3 mM dithiothreitol. For some experiments, untagged full-length recombinant

Bdp1 was utilized that was custom-expressed in bacteria and purified by Genscript.

Electrophoretic Mobility Shift Assays (EMSAs)

EMSAs were performed as previously described (Hung and Stumph, 2012).

Briefly, reactions were carried out in 21-ul volumes in a final concentration of 100 mM KCl, 25 mM HEPES K+ (pH 7.6), 12.5 mM MgCl2, 10 µM ZnCl2, 100 µM

EDTA (pH 8.0) and 10% glycerol. Each reaction also contained 2 µg of poly (dG-dC)

(Sigma #P9389). 32P-labeled DNA probes (described in the Results and figure legends) were incubated for 30 min at 25C. For antibody-induced supershifts, the antibodies were added halfway through the 30 min incubation. Complexes were then run on 4% non-denaturing polyacrylamide gels in 25 mM Tris, 190 mM glycine, 1 mM EDTA (pH 8.3) and detected by autoradiography.

RESULTS

In recent work, this lab found that Drosophila TFIIIB, consisting of TBP, Brf1, and Bdp1, could be assembled in vitro on the TATA box of the U6:96Ab snRNA gene promoter independently of the presence of DmSNAPc (Kang et al., 2016). However, mutation of the DmSNAPc binding site (the PSEA) resulted in the inactivation of U6 transcription in cells (Lai et al., 2005), suggesting that recruitment of TFIIIB depends upon DmSNAPc in vivo. To better understand the biochemical mechanisms behind this recruitment, we decided to investigate whether interactions between DmSNAPc and TFIIIB could be identified on the U6 promoter in vitro.

Bdp1 can be recruited to the U6 promoter by DmSNAPc – We first investigated whether interactions between DmSNAPc and each of the TFIIIB subunits could be detected by EMSAs. His6-tagged proteins (TBP, Brf1, or DmSNAPc [DmSNAP43,

DmSNAP50, and DmSNAP190 together]) were overexpressed in Drosophila S2 cells and partially purified by nickel-chelate chromatography as described in Materials and

Methods. Bdp1 was expressed as a tagged protein in S2 cells or was obtained in an untagged form after expression in E. coli. The DNA EMSA probe contained sequences extending from positions –75 to -7 relative to the U6:96Ab gene transcription start site.

Figure 1: Assembly of DmSNAPc with individual subunits of TFIIIB on the U6 snRNA gene promoter. A, Autoradiogram of an EMSA performed with a 32P-labeled DNA probe that contained U6 promoter DNA from position -7 to -75 base pairs upstream of the transcription start site. Reactions shown in lanes 4-7, 11-14, and 18-20 each contained 4 µl of DmSNAPc. Reactions run in lanes 1-3 and 4-6 contained TBP in increasing amounts (1, 1.7, or 2.5 µl) as indicated above each three-lane set. Lanes 8-10 and 11-13 contained Brf1 in increasing amounts (1, 2, or 4 µl) as indicated above the respective lanes. (The Brf1 was active as indicated by its ability to form Brf1-TBP complexes and Brf1- Bdp1-TBP complexes (Kang et al., 2016) and [not shown]). Lanes 15-17 and 18-20 contained recombinant Bdp1 in increasing amounts (25, 50, or 100 ng). B, EMSA super-shifts with antibodies specific for DmSNAPc and (Myc-tagged)-Bdp1. Reactions shown in each lane contained 1.5 µl of DmSNAPc, while those in lanes 2-4 each additionally contained 4.5 µl Bdp1. Lanes 3 and 4 contained 1.5 µl of either anti-Myc antibody or anti-DmSNAP190 antibody respectively. Lane 1 is from the same gel and length of autoradiogram exposure as lanes 2-4. Free probe was run off the gel to obtain better resolution between shifted bands.

Fig. 1A shows that TBP (lanes 1-3) and DmSNAPc (lanes 7 and 14) each bound individually to the DNA probe. This was expected since the probe contained both a PSEA (-63 to -43) and a TATA box (-30 to -23). (Despite the significant difference in molecular size, the TBP band ran just barely ahead of the SNAPc band, probably due to the bending of the DNA by TBP.) When DmSNAPc and TBP were both added to the reactions (lanes 4-6), the TBP band no longer appeared. Instead, bands of slower mobility were now seen, and the amount of free probe remaining was drastically reduced. This suggests that there was at least a certain degree of cooperative binding between DmSNAPc and TBP to the DNA fragment. This is

36 similar to the findings of others who observed fairly weak cooperative binding of human TBP and SNAPc to a human U6 gene promoter that was considerably strengthened by the addition of Brf2 (Hinkley et al., 2003; Ma and Hernandez, 2002;

Saxena et al., 2005).

Brf1, on the other hand, did not bind to DNA by itself, nor was there any evidence that it could stably interact with DmSNAPc on the DNA (Fig. 1A, lanes 8-

13). As expected, Bdp1 alone did not bind to DNA (lanes 15-17), but surprisingly, the bacterially-expressed Bdp1 stably interacted with DmSNAPc to produce a super- shifted band corresponding to a Bdp1-DmSNAPc complex (lanes 18-20). To verify that the super-shifted band contained both DmSNAPc and Bdp1, we expressed Bdp1 with a C-terminal Myc tag in S2 cells. When the tagged Bdp1 was added to an EMSA reaction that contained DmSNAPc, the same Bdp1-DmSNAPc super-shifted band was observed as previously seen with the bacterially-expressed Bdp1 (Fig. 1B, lane 2, and data not shown). This band was further super-shifted by the addition of either anti-

Myc antibody against tagged Bdp1 or anti-DmSNAP190 antibody. These results reveal that DmSNAPc and Bdp1 can form a stable complex on the U6 promoter even though Bdp1 has no detectable DNA binding activity of its own.

Recruitment of Bdp1 to the U6 promoter by DmSNAPc involves a region of Bdp1 just C-terminal of the Bdp1 SANT domain – We next employed two series of truncation mutations within Bdp1 to map a region of Bdp1 required for its recruitment by DmSNAPc. The most prominent feature of Bdp1 is that it contains an evolutionarily conserved SANT domain that extends between amino acids 346 and

403 in the D. melanogaster protein (Fig. 2A, top rectangle). In yeast, the Bdp1 SANT domain is involved in interacting with Brf1 (Kassavetis et al., 2006; Saida, 2008). The truncation constructs shown in Fig. 2A were expressed in S2 cells, subjected to purification by nickel chelate chromatography, and used in EMSAs with wild type

DmSNAPc (Fig. 2B).

Figure 2: A region of Bdp1 C-terminal of the SANT domain is required for its recruitment by DmSNAPc. A, Truncation constructs used to map a region of Bdp1 required for its interaction with DmSNAPc on the U6 promoter. The top rectangle represents full-length (FL) fly Bdp1 (695 amino acid residues). The location of the highly-conserved SANT domain is also indicated. The black bars beneath represent the truncation constructs utilized in EMSA reactions. All constructs were expressed in S2 cells and were tagged with Flag-Myc-His6 epitopes at the C-terminus. The numbers at each end of the bars indicate the extent of the wild type amino acid residues present in the expressed constructs. B, EMSAs showing the ability of the full length and truncated Bdp1 constructs to assemble with DmSNAPc on U6 promoter DNA. The location of the shifted bands and free probe are indicated to the left of the panel. The amount of construct added in each reaction was adjusted so that the intensity of the signal obtained from the shifted bands was similar in each lane and varied between 1.5 µl and 3.0 µl in the left panel and between 3 µl and 5 µl in the right panel.

Each of the Bdp1 constructs of the N-terminal truncation series (constructs A through D) formed a slower-migrating complex on the DNA when added together with DmSNAPc (Fig. 2B, lanes 3-6). These data revealed that amino acid residues C- terminal of position 424 were sufficient for Bdp1 recruitment by DmSNAPc.

Furthermore, the data indicated that the SANT domain was not required for Bdp1 recruitment.

When the reciprocal set of C-terminal Bdp1 truncation constructs was used in the EMSAs, the constructs that contained the first 615 or 510 residues from the N- terminus of the protein (constructs E and F) formed a complex with DmSNAPc on the

U6 promoter fragment (Fig. 2B, lanes 9 and 10). However, when Bdp1 was truncated from the C-terminus leaving only 464 N-terminal amino acid residues or fewer remaining (constructs G, H, and I), no slower-migrating complex was observed (lanes

11-13). Together these results implicated a region of Bdp1 just C-terminal of the

SANT domain (i.e., between residues 424 and 510) as being required for Bdp1 recruitment by DmSNAPc.

Bdp1 recruitment by DmSNAPc is dependent upon a U6 PSEA versus a U1 PSEA –

We have previously shown that the PSEAs of Drosophila U1 and U6 genes are not functionally interchangeable for snRNA transcription even though they differ at only 5 of 21 nucleotide positions and are both recognized and bound by DmSNAPc. The U1

PSEA is only able to recruit Pol II, and the U6 PSEA is only capable of recruiting Pol

III, as exchange of the PSEAs inactivated snRNA expression in vivo and switched the polymerase specificity in vitro (Barakat and Stumph, 2008; Hung and Stumph, 2011;

Jensen et al., 1998; Lai et al., 2005; McNamara-Schroeder et al., 2001). Furthermore, evidence accumulated by site-specific protein-DNA photo-cross-linking has strongly suggested that DmSNAPc binds in different conformations to the U1 and U6 PSEAs

(Hung and Stumph, 2011; Kang et al., 2014; Kim et al., 2010a, b; Lai et al., 2005; Li et al., 2004; Wang and Stumph, 1998).

Figure 3: A Pol III-specific PSEA is necessary and sufficient for Bdp1 recruitment by DmSNAPc. A, The five nucleotide differences between the U6 and U1 PSEA sequences used for the EMSAs in (B) are indicated by asterisks. B, Increasing amounts of Bdp1 were added to the reactions containing a constant amount of DmSNAPc with either wild-type U6 promoter DNA (lanes 1-7) or with a probe (Lanes 8-14) that was identical except that it contained five nucleotide changes that converted the U6 PSEA to a U1 PSEA. The panel to the right shows a reciprocal experiment which employed a wild-type U1 promoter DNA probe (sequences from -75 to -7 relative to the U1 transcription start site) (lanes 15- 21) or a U1 probe in which the U1 PSEA was switched to a U6 PSEA (lanes 22-28). Lanes 2-8, 9-14, 16-21, and 23-28 contained bacterially-expressed Bdp1 in increasing amounts as follows: 12.5, 25, 50, 75, 100, and 200 ng.

To further investigate the molecular mechanism of the RNA polymerase

40 specificity of snRNA gene promoters, we investigated whether the ability of

DmSNAPc to recruit Bdp1 is dependent upon a U6 PSEA (that specifically recruits

Pol III) versus a U1 PSEA (that recruits Pol II). To do this, EMSAs were performed utilizing a DNA probe that was identical to the wild type U6 EMSA probe except that it contained 5 base changes that converted the U6 PSEA to a U1 PSEA (Fig. 3A). Fig.

3B shows that when DmSNAPc bound to the wild type U6 probe, the addition of increasing amounts of Bdp1 resulted in the formation of a DmSNAPc-Bdp1 complex on the DNA (lanes 1-7). In contrast, when the probe was used that contained the U1

PSEA in the context of the U6 promoter, DmSNAPc itself bound very efficiently to the U1 PSEA but astonishingly was unable to recruit Bdp1 (lanes 8-14).

As confirmation of the above results, we next performed the reciprocal experiment in which 5 base changes were made in the U1 promoter that converted the

U1 PSEA to a U6 PSEA. As expected from the preceding result, when the DNA probe contained entirely wild type U1 promoter sequences, DmSNAPc bound to the DNA but was unable to recruit Bdp1 (Fig. 3, lanes 15-21.) However, when five base changes were made that switched the U1 PSEA to a U6 PSEA in the context of the U1 promoter, DmSNAPc successfully recruited Bdp1 (lanes 22-28). These results support the concept that the sequence of the PSEA plays a role in Bdp1 recruitment by differentially affecting the conformation of DmSNAPc, thereby enabling DmSNAPc to recruit Bdp1 to the U6 promoter but not to the U1 promoter.

The DmSNAPc-Bdp1 complex can recruit TBP to the U6 promoter - Because the

DmSNAPc-mediated recruitment of Bdp1 to the U6 promoter was a novel

41 observation, we next examined whether the DmSNAPc-Bdp1 complex might play a role in the recruitment of TBP to the U6 promoter DNA. For the EMSA experiments shown in Fig. 4A, each set of three lanes (i.e., lanes 3-5, 6-8, and 9-11) contained the same gradually increasing amounts of TBP. Two lanes (12 and 13) contained still higher amounts of TBP without any other proteins added. When TBP alone was present in increasing amounts in the reactions (lanes 9-13), very weak band-shifts due to TBP were observed only with the higher amounts of TBP (lanes 11-13). When the same amounts of TBP used in lanes 9-11 were added to a constant amount of

DmSNAPc (Fig. 4A, lanes 3-5), a light smear of bands was observed that likely corresponded to DNA fragments co-occupied by DmSNAPc and TBP (as seen more clearly in Fig. 1A [lanes 4-6], which used higher amounts of TBP). However, when

Bdp1 was present together with DmSNAPc, even the lowest amount of TBP was sufficient to form a strong higher-order complex of slower mobility (lanes 6-8).

Moreover, essentially all the DmSNAPc band-shift was translated into the higher order complex. These results suggest that there is strong cooperative binding of

DmSNAPc, Bdp1, and TBP to the U6 promoter.

The presence of all three proteins in the higher order complex was confirmed by antibody supershifts (Fig. 4B). The TBP-Bdp1-DmSNAPc complex migrated more slowly than the Bdp1-DmSNAPc complex (lane 3 vs. lane 2), and this complex was further super-shifted by antibodies against either DmSNAP190, the Myc epitope on

Bdp1, or the V5 epitope on TBP (Fig. 4B, lanes, 4, 5, and 6 respectively). The above experiments revealed that DmSNAPc and Bdp1 together were capable of recruiting

TBP to the wild type U6 promoter.

Figure 4: The DmSNAPc-Bdp1 complex efficiently recruits TBP to the U6 promoter. A, EMSA reactions with a wild type U6 promoter DNA probe and increasing amounts of TBP (0.25, 0.5, and 0.75 µl) added either alone (lanes 9-11), in the presence of DmSNAPc (lanes 3-5), or in the presence of both DmSNAPc and Bdp1 (lanes 6-8). B, EMSAs with specific antibody supershifts are shown. Lanes 1 and 2 are each from the same gel as lanes 3-6, although non-adjacent. Each reaction contained 3 µl DmSNAPc; lanes 2-6 each contained 2.5 µl of Bdp1; and lanes 3-6 each contained 0.5 µl of TBP. The TBP-Bdp1-DmSNAPc complex (lane 3) was supershifted by antibodies against either DmSNAPc (α- 190, lane 4), Bdp1 (α-Myc, lane 5), or TBP (α-V5, lane 6). Free probe was run off the gel to improve the resolution among the shifted bands.

TBP recruitment by the DmSNAPc-Bdp1 complex requires a TATA box – We next investigated whether TBP recruitment under such circumstances was also dependent upon the presence of the TATA box in the U6 promoter. For this, a DNA EMSA probe was used that was identical to the wild type probe except the TATA sequence

(TTTATATA) was mutated to GGGACCTC. As expected, TBP by itself was able to bind to the wild type probe but did not bind to the mutant TATA probe (Fig. 5, lanes 1 and 2). On the other hand, DmSNAPc bound equally well to both the wild type and mutant TATA probes (lanes 3 and 4). Furthermore, the mutation of the TATA sequence had no effect on the recruitment of Bdp1 by DmSNAPc (Fig 5, lanes 5 and

6). In stark contrast, the DmSNAPc-Bdp1 complex effectively recruited TBP to the wild type promoter but was unable to recruit TBP to the mutant TATA promoter

(compare lanes 7 and 8). This result indicated that the recruitment of TBP to the U6 promoter was dependent upon the presence of a TATA sequence.

Figure 5: A TATA box is required for recruitment of TBP to the U6 promoter by DmSNAPc- Bdp1. EMSAs are shown that utilized either the wild type U6 promoter probe or a DNA fragment that was identical except for a mutated TATA box. Various combinations of DmSNAPc (3 µl), Bdp1 (2 µl) and TBP (1 µl in lanes 1 and 2 and 0.4 µl in lanes 7 and 8) were incubated with the two different U6 promoter fragments as indicated above the lanes.

A region of Bdp1 that participates in TBP recruitment is close to or overlaps with the region of Bdp1 required for interaction with DmSNAPc – We next investigated whether a specific region of Bdp1 is involved in recruiting TBP to the U6 promoter.

For this purpose, we utilized the truncation mutations of Bdp1 that were able to assemble with DmSNAPc on the U6 promoter (Fig. 2). Fig. 6 shows that each of these

Bdp1 truncation mutants was able to recruit TBP to form a higher order complex on

44 the U6 promoter. This indicated that a region of Bdp1 located between residues 424 and 510 is important for TBP recruitment to the U6 promoter. Thus, at this level of resolution, an 87-amino-acid region of Bdp1 plays a role in both the recruitment of

Bdp1 by DmSNAPc and in the recruitment of TBP by Bdp1.

Figure 6: Mapping a region of Bdp1 that participates in the recruitment of TBP by the DmSNAPc-Bdp1 complex on U6 promoter DNA. EMSAs utilizing various Bdp1 truncation constructs (diagrammed in Fig. 2) are shown. The particular Bdp1 truncation construct utilized in each lane is indicated in the top line of the figure and varied between 2 µl and 7 µl. Reactions run in all lanes contained DmSNAPc (3 µl); TBP (0.5 µl) was added to the reactions run in all the even-numbered lanes. The asterisks indicate the positions of the bands formed by DmSNAPc and the different Bdp1 constructs in the absence of TBP. In all cases, the DNA probe was a wild type U6 promoter DNA fragment.

We did notice a consistent reduction in the intensity of the TBP-Bdp1-

DmSNAPc band in the case of construct “D” which lacked the SANT domain but contained residues C-terminal of position 424 (Fig. 6, lane 12). (Note that the Bdp1-

SNAPc bands, denoted by asterisks, are of similar intensities in lanes 9 and 11, but less of these bands are further super-shifted by TBP in lanes 10 and 12 respectively.)

Thus, the Bdp1 SANT domain may also contribute to the ability of the DmSNAPc-

Bdp1 complex to recruit TBP.

DISCUSSION

Considerable progress has been made through studies in the human system toward understanding how SNAPc and TFIIIB form a stable complex on U6 snRNA gene promoters, but much remains unknown. SNAPc was found to stabilize the binding of TBP to the U6 promoter TATA box by means of cooperative binding interactions (Hinkley et al., 2003; Ma and Hernandez, 2002; Mittal and Hernandez,

1997; Saxena et al., 2005). We likewise observed weak cooperative binding of

DmSNAPc and TBP to U6 promoter DNA (Fig. 1). Interestingly, we also detected an interaction between promoter-bound DmSNAPc and Bdp1, an interaction to our knowledge not previously reported. This is particularly significant in that Bdp1 possessed no DNA binding activity of its own. By truncation experiments, a region of

Bdp1 required for recruitment by DmSNAPc was localized to a region of Drosophila

Bdp1 between residues 424 and 510, just C-terminal of the conserved SANT domain

(Fig. 2).

Furthermore, we found that the DmSNAPc-Bdp1 complex was able to recruit

TBP to the TATA box with much greater efficiency than DmSNAPc alone (Fig. 4A).

Thus, Bdp1 apparently acts as a bridge between DmSNAPc and TBP to establish a quaternary complex of the three proteins tightly bound to the U6 promoter DNA.

Experiments with the Bdp1 truncation constructs suggest that the region of Bdp1 just

C-terminal of its SANT domain is required not only for its recruitment by DmSNAPc but is likely also a region of interaction with TBP.

Figure 7: Bdp1 is required, together with DmSNAPc bound to a Pol III-specific PSEA and TBP bound to a TATA box, to form a stable protein-DNA complex on the bipartite Drosophila U6 snRNA gene promoter. A, The wild type scenario for a fly Pol III-transcribed snRNA gene is shown. DmSNAPc binds to a U6 PSEA in a conformation that allows it to recruit Bdp1 and hence TBP. B, Mutation of the TATA box interferes with TBP recruitment but does not affect Bdp1 recruitment by DmSNAPc. C, Switching the U6 PSEA to a U1 PSEA alters the conformation of DmSNAPc and interferes with its ability to recruit Bdp1. In each of the diagrams, the components of DmSNAPc as well as Bdp1 are placed on the promoter sequences based upon site-specific protein-DNA photo-cross- linking experiments (Hung and Stumph, 2011; Kang et al., 2014; Kim et al., 2010a, b; Lai et al., 2005; Li et al., 2004; Wang and Stumph, 1998).

Perhaps most interestingly, we found that DmSNAPc must be bound to a U6

PSEA in order to recruit Bdp1. A 5-base-pair change that converted the U6 PSEA to a

U1 PSEA in the otherwise complete context of the U6 promoter abolished the ability of bound DmSNAPc to recruit Bdp1, even though DmSNAPc was still efficiently bound. This suggests that, when DmSNAPc is bound to a U1 PSEA, the Bdp1-

47 interaction surface of DmSNAPc is occluded or wrongly disposed to interact with

Bdp1. This finding provides new evidence to support a model of snRNA gene RNA polymerase specificity previously proposed by our lab (Hung and Stumph, 2011; Kang et al., 2014; Kim et al., 2010a, b; Li et al., 2004; Wang and Stumph, 1998) in which the specific DNA sequence of the PSEA acts as an allosteric effector of DmSNAPc conformation that determines its ability to recruit only Pol III general transcription factors to the U6 gene and Pol II general transcription factors to the U1 gene.

Certain aspects of this model are represented schematically in Fig. 7. When

DmSNAPc binds to a U6 promoter, it exists in a conformation whereby it can recruit

Bdp1 and subsequently TBP for Pol III transcription (Fig. 7A). Two alternatives are also shown that would prevent Pol III from being recruited to Pol II-specific snRNA promoters, which lack TATA boxes and contain functionally distinct PSEAs. If the promoter contained a U6 PSEA but no TATA box, Bdp1 could be recruited but not

TBP (Fig. 7B). If the promoter contained a U1 PSEA instead of a U6 PSEA, Bdp1 could not be recruited and hence TBP also would not be recruited (Fig. 7C).

Altogether, our results indicate that a network of very specific molecular interactions must occur to cooperatively establish a stable protein-DNA complex on the U6 promoter. This interaction network must involve at least five important conserved components in flies: a Pol III-specific PSEA, DmSNAPc (while bound to a Pol III- specific PSEA), Bdp1, TBP, and the TATA box (Fig. 7A).

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Barakat, N.H., and Stumph, W.E. (2008). TBP recruitment to the U1 snRNA gene promoter is disrupted by substituting a U6 proximal sequence element A (PSEA) for the U1 PSEA. FEBS Lett 582, 2413-2416.

Canella, D., Praz, V., Reina, J.H., Cousin, P., and Hernandez, N. (2010). Defining the RNA polymerase III transcriptome: Genome-wide localization of the RNA polymerase III transcription machinery in human cells. Genome Res 20, 710-721.

Carbon, P., and Krol, A. (1991). Transcription of the Xenopus laevis selenocysteine tRNA(Ser)Sec gene: a system that combines an internal B box and upstream elements also found in U6 snRNA genes. EMBO J 10, 599-606.

Egloff, S., O'Reilly, D., and Murphy, S. (2008). Expression of human snRNA genes from beginning to end. Biochem Soc Trans 36, 590-594.

Geiduschek, E.P., and Tocchini-Valentini, G.P. (1988). Transcription by RNA polymerase III. Annu Rev Biochem 57, 873-914.

Henry, R.W., Ford, E., Mital, R., Mittal, V., and Hernandez, N. (1998). Crossing the line between RNA polymerases: Transcription of human snRNA genes by RNA polymerases II and III. Cold Spring Harbor SympQuantBiol 63, 111-120.

Henry, R.W., Sadowski, C.L., Kobayashi, R., and Hernandez, N. (1995). A TBP-TAF complex required for transcription of human snRNA genes by RNA polymerases II and III. Nature 374, 653-656.

Hinkley, C.S., Hirsch, H.A., Gu, L.P., LaMere, B., and Henry, R.W. (2003). The small nuclear RNA-activating protein 190 Myb DNA binding domain stimulates TATA box-binding protein-TATA box recognition. J Biol Chem 278, 18649-18657.

Hung, K.H., and Stumph, W.E. (2011). Regulation of snRNA gene expression by the Drosophila melanogaster small nuclear RNA activating protein complex (DmSNAPc). Crit Rev Biochem Mol Biol 46, 11-26.

Hung, K.H., and Stumph, W.E. (2012). Localization of residues in a novel DNA- binding domain of DmSNAP43 required for DmSNAPc DNA-binding activity. FEBS Lett 586, 841-846.

Hung, K.H., Titus, M., Chiang, S.C., and Stumph, W.E. (2009). A map of Drosophila melanogaster small nuclear RNA-activating protein complex (DmSNAPc) domains involved in subunit assembly and DNA binding. J Biol Chem 284, 22568-22579.

Isogai, Y., Takada, S., Tjian, R., and Keles, S. (2007). Novel TRF1/BRF target genes revealed by genome-wide analysis of Drosophila Pol III transcription. Embo J 26, 79- 89.

Jawdekar, G.W., and Henry, R.W. (2008). Transcriptional regulation of human small nuclear RNA genes. Biochim Biophys Acta 1779, 295-305.

Jensen, R.C., Wang, Y., Hardin, S.B., and Stumph, W.E. (1998). The proximal sequence element (PSE) plays a major role in establishing the RNA polymerase specificity of Drosophila U-snRNA genes. Nucleic Acids Res 26, 616-622.

Kang, J.J., Kang, Y.S., and Stumph, W.E. (2016). TFIIIB subunit locations on U6 gene promoter DNA mapped by site-specific protein-DNA photo-cross-linking. FEBS Lett 590, 1488-1497.

Kang, Y.S., Kurano, M., and Stumph, W.E. (2014). The Myb domain of the largest subunit of SNAPc adopts different architectural configurations on U1 and U6 snRNA gene promoter sequences. Nucleic Acids Res 42, 12440-12454.

Kassavetis, G.A., Driscoll, R., and Geiduschek, E.P. (2006). Mapping the principal interaction site of the Brf1 and Bdp1 subunits of Saccharomyces cerevisiae TFIIIB. J Biol Chem 281, 14321-14329.

Kim, M.K., Kang, Y.S., Lai, H.T., Barakat, N.H., Magante, D., and Stumph, W.E. (2010b). Identification of SNAPc subunit domains that interact with specific nucleotide positions in the U1 and U6 gene promoters. Mol Cell Biol 30, 5257.

Kunkel, G.R., Maser, R.L., Calvet, J.P., and Pederson, T. (1986). U6 small nuclear RNA is transcribed by RNA polymerase III. ProcNatlAcadSciUSA 83, 8575-8579.

Lai, H.T., Chen, H., Li, C., McNamara-Schroeder, K.J., and Stumph, W.E. (2005). The PSEA promoter element of the Drosophila U1 snRNA gene is sufficient to bring DmSNAPc into contact with 20 base pairs of downstream DNA. Nucleic Acids Res 33, 6579-6586.

Li, C., Harding, G.A., Parise, J., McNamara-Schroeder, K.J., and Stumph, W.E. (2004). Architectural arrangement of cloned proximal sequence element-binding

50 protein subunits on Drosophila U1 and U6 snRNA gene promoters. Mol Cell Biol 24, 1897-1906.

Ma, B.C., and Hernandez, N. (2002). Redundant cooperative interactions for assembly of a human U6 transcription initiation complex. Mol Cell Biol 22, 8067-8078.

McNamara-Schroeder, K.J., Hennessey, R.F., Harding, G.A., Jensen, R.C., and Stumph, W.E. (2001). The Drosophila U1 and U6 gene proximal sequence elements act as important determinants of the RNA polymerase specificity of snRNA gene promoters in vitro and in vivo. J Biol Chem 276, 31786-31792.

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Sadowski, C.L., Henry, R.W., Lobo, S.M., and Hernandez, N. (1993). Targeting TBP to a non-TATA box cis-regulatory element: a TBP-containing complex activates transcription from snRNA promoters through the PSE. Genes Dev 7, 1535-1548.

Saida, F. (2008). Structural characterization of the interaction between TFIIIB components Bdp1 and Brf1. Biochemistry 47, 13197-13206.

Saxena, A., Ma, B., Schramm, L., and Hernandez, N. (2005). Structure-function analysis of the human TFIIB-related factor II protein reveals an essential role for the C-terminal domain in RNA polymerase III transcription. Mol Cell Biol 25, 9406-9418.

Schramm, L., and Hernandez, N. (2002). Recruitment of RNA polymerase III to its target promoters. Genes Dev 16, 2593-2620.

Schramm, L., Pendergrast, P.S., Sun, Y.L., and Hernandez, N. (2000). Different human TFIIIB activities direct RNA polymerase III transcription from TATA- containing and TATA-less promoters. Genes Dev 14, 2650-2663.

Teichmann, M., Wang, Z.X., and Roeder, R.G. (2000). A stable complex of a novel transcription factor IIB-related factor, human TFIIIB50, and associated proteins mediate selective transcription by RNA polymerase III of genes with upstream promoter elements. Proc Natl Acad Sci USA 97, 14200-14205.

Verma, N., Hung, K.H., Kang, J.J., Barakat, N.H., and Stumph, W.E. (2013). Differential utilization of TATA box-binding protein (TBP) and TBP-related factor 1 (TRF1) at different classes of RNA polymerase III promoters. J Biol Chem 288, 27564-27570.

Wang, Y., and Stumph, W.E. (1998). Identification and topological arrangement of Drosophila proximal sequence element (PSE)-binding protein subunits that contact the PSEs of U1 and U6 snRNA genes. Mol Cell Biol 18, 1570-1579.

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Yoon, J.B., Murphy, S., Bai, L., Wang, Z., and Roeder, R.G. (1995). Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III- dependent small nuclear RNA genes. Mol Cell Biol 15, 2019-2027.

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ACKNOWLEDGEMENTS: This Chapter is written as a manuscript to be submitted for publication immediately following approval of this thesis. SNAPc interacts with Bdp1 to establish a stable protein-DNA complex with TBP on a U6 snRNA gene promoter. The dissertation author is the primary author of this manuscript. The Bdp1 constructs were

52 generated by Yoon Soon Kang and Ann Marie Hurlburt, whereas the EMSAs utilizing

Bdp1 constructs in absence or presence of TBP, and mutated TATA probe were performed by Ann Marie Hurlburt. Nickel-chelate chromatography of many of the proteins was done by Phuc Phan. All other data in this paper were produced by this dissertation author.

CHAPTER 3

Experiments to map a region of DmSNAPc responsible for recruitment of Bdp1 to the

Drosophila melanogaster U6 snRNA gene promoter

53 54

INTRODUCTION

The Drosophila melanogaster SNAPc (DmSNAPc) is a hetero-trimeric transcription factor [Lai et al., 2008] that is required for the synthesis of the U1, U2,

U4, U5, and U6 small nuclear RNAs (snRNAs) [Su et al., 1997, Li et al., 2004;

Barakat and Stumph, 2008; Hernandez et al., 2007]. DmSNAPc binds sequence- specifically to the conserved ~21 base pair (bp) promoter element termed the PSEA

(See Figs. 1 and 2 in the General Introduction) [Hernandez et al., 2007] and can activate transcription of the Drosophila U1 and U6 snRNA genes in vitro [Su et al.,

1997]. Our lab also showed that DmSNAPc contains three distinct polypeptides

(DmSNAP43, DmSNAP50, and DmSNAP190) each of which contacts the DNA

[Wang and Stumph, 1998]. Moreover, the face of the DNA helix that each of the subunits interacts with (relative to the PSEA sequence and to each other) was determined [Wang and Stumph, 1998]. It was found that DmSNAP190 contacts the entire length of the PSEA while DmSNAP43 and DmSNAP50 interact only with the

3’ end of the PSEA. In addition, DmSNAP43 cross-links to DNA up to 20 bp downstream of a U1 PSEA, but only 5 bp downstream of a U6 PSEA. Furthermore, the in vitro and in vivo transcription data of our lab indicate that these conformational differences are differentially interpreted by the basal transcription machinery to confer promoter specificity for either Pol II or Pol III (See Fig. 3 in the General Introduction).

The data shown in Chapter 2 reveals a novel interaction between DmSNAPc and Bdp1 on the U6 snRNA gene promoter. Here, I wanted to investigate the regions of DmSNAPc involved in this interaction. Since DmSNAP43 cross-links to the DNA furthest downstream of the U6 PSEA [Li et al., 2004; Lai et al., 2005], it is perhaps the most attractive candidate that could be involved in DmSNAPc co-operative binding with Bdp1 on the U6 snRNA gene. Previous studies in our lab utilized a variety of mutant constructs to identify the DmSNAPc subunit domains involved in subunit assembly and DNA-binding [Hung et al., 2009; Hung and Stumph, 2012]. I employed some of those existing constructs to investigate subunit domains involved in the co- operativity between DmSNAPc and Bdp1.

One truncation construct of DmSNAP43 was first used (Fig. 2A). N- or C- terminal truncations that remove more amino acids than in this construct eliminated the DNA-binding activity of DmSNAPc [Hung et al., 2009; Hung and Stumph, 2012] and could therefore not be used for my experiments. Besides this truncation construct, the lab also had on hand a small library of alanine-scanning constructs that stretch between residues 193-272 of DmSNAP43 (Fig. 1B). These mutations are within the domain of DmSNAP43 that lies farthest in the downstream direction (Fig. 1C) and closest to the TFIIIB binding site. Thus, we believed this domain of DmSNAP43

(residues 193-272) had an increased potential for interaction with Bdp1.

A 2 8 155 363 DmSNAP43(WT) N C

2 274 DmSNAP43(2-274) N C

Figure 1: Schematic representation of various DmSNAP43 constructs

(A) Schematic diagram of full-length DmSNAP43 and a truncation construct that did not eliminate the DNA-binding activity of DmSNAPc. (B) Various DmSNAP43 alanine substitution constructs. Mutant 1 and mutants 4 through 14 were used for EMSAs with Bdp1. Mutants 2 and 3 resulted in a loss of DNA-binding activity and were therefore not used in my assays. (C) Schematic representation of DmSNAP43 domains that contact DNA when DmSNAPc binds to a U6 PSEA [Kim et al., 2010; Hung and Stumph, 2011].

MATERIALS AND METHODS

The preparation of untagged constructs encoding wild type DmSNAP50 and

DmSNAP190 and N-terminal 6xHis-FLAG-tagged DmSNAP43 constructs under the control of the copper-inducible metallothionein promoter have been previously described in Chapter 2. Each of the full-length and mutant DmSNAP43 constructs used in these experiments had 6xHis-FLAG tags at their N-terminus. Alanine- substitution constructs of DmSNAP43 (shown in Fig 1B) were prepared by site- directed mutagenesis as previously described [Hung and Stumph, 2012]. Expression plasmids for tagged wild type or mutant DmSNAP43 were used to co-transfect S2 cells with untagged DmSNAP50 and DmSNAP190 [Hung et al., 2009]. Various wild type or alanine-scanning mutants were purified using nickel chelate chromatography as described in Chapter 2. The purified proteins were then employed in EMSAs utilizing the wild type U6 promoter probe as described in Chapter 2.

Figure 2: Mapping a region of DmSNAPc involved in interaction with Bdp1 on U6 promoter DNA. EMSAs showing the ability of the full length and various DmSNAP43 constructs (diagrammed in Fig. 2A and 2B) to recruit Bdp1 to the U6 promoter DNA. The particular DmSNAP43 construct (full length, alanine-scanning mutation or truncation) utilized in each lane is indicated in the top line of the figure. Lanes 25-30 are each from the same gel, although non-adjacent. The amount of construct added in each reaction was adjusted so that the intensity of the signal obtained from the shifted bands without Bdp1 was similar in each lane and varied between 2.5 ul and 10 ul.

RESULTS AND DISCUSSION

To map the regions of DmSNAPc involved in the interactions with Bdp1, various stably transfected S2 cell lines co-overexpressing tagged truncated or alanine- scanning mutant constructs of DmSNAP43 with wild type DmSNAP190 and

DmSNAP50 were created, and proteins were purified by nickel-chelate chromatography following copper induction. These proteins were then utilized in

EMSAs in which the reactions contained either SNAPc (full length or truncated or mutated) alone (in odd numbered lanes of Fig. 2) or both SNAPc and Bdp1 (in even numbered lanes of Fig. 2) along with the wild type U6 promoter probe. Since the alanine-scanning mutations lie within the domain of DmSNAP43 that lies farthest in the downstream direction (Fig. 1C), we suspected that this domain was a good candidate for interaction with Bdp1.

However, the reactions containing either the full length DmSNAPc (Fig. 2, lanes 1, 2, 13, 14, and 29-32) or various alanine-scanning mutations of DmSNAP43

(lanes 3-12, and 15-28) or the C-terminal truncation of DmSNAP43 (lanes 33 and 34) still retained an ability to recruit Bdp1 (Fig 3, all even numbered lanes). The results with mutant 1 shown in lanes 3 and 4 initially suggested that mutations within the conserved Cluster 1 might interfere with Bdp1 recruitment. However, subsequent repetitions of this reaction (not shown) indicated that even mutant 1 retained significant ability to recruit Bdp1.

In summary, the results indicated that the DmSNAP43 regions we studied here are not essential for the interaction of DmSNAPc with Bdp1. However, they also do

60 not rule out the possibility that one or more of these regions could be involved in interacting with DmSNAPc. There may be redundancy in the interactions that are not uncovered in these assays. For example, more than one of the assayed regions of

DmSNAP43 could participate in Bdp1 recruitment, and it is quite possible that one or more of the other subunits of DmSNAPc could also be involved. The lab has two

DmSNAP190 truncation constructs that retain DNA-binding activity when complexed with DmSNAP50 and DmSNAP43; these could be considered for further investigating the region of SNAPc that interacts with Bdp1.

REFERENCES

Hernandez, G., Valafar, F., and Stumph, W.E. (2007). Insect small nuclear RNA gene promoters evolve rapidly yet retain conserved features involved in determining promoter activity and RNA polymerase specificity. Nucleic Acids Res. 35, 21-34.

Hung, K. H., and Stumph, W. E. (2012). Localization of residues in a novel DNA- binding domain of DmSNAP43 required for DmSNAPc DNA-binding activity. FEBS Letters, 586.6, 841-846.

Hung, K. H., Titus, M., Chiang, S. C., and Stumph, W. E. (2009). A map of Drosophila melanogaster small nuclear RNA-activating protein complex (DmSNAPc) domains involved in subunit assembly and DNA binding. J. Biol. Chem. 284, 22568- 22579.

Li, C., G.A. Harding, J. Parise, K.J. McNamara-Schroeder, and W.E. Stumph. (2004). Architectural arrangement of cloned proximal sequence element-binding proein subunits on Drosophila U1 and U6 snRNA gene promoters. Mol Cell Biol 24: 1897- 1906.

Su, Y., Song, Y., Wang, Y., Jessop, L., Zhan, L.C. and Stumph, W.E. (1997). Characterization of a Drosophila proximal-sequence-element-binding protein involved in transcription of small nuclear RNA genes. Eur. J. Biochem. 248, 231-237.

ACKNOWLEDGEMENTS: The data in this Chapter are not currently intended to be submitted for publication. They may be submitted as part of a future publication. The DmSNAP43 constructs were provided by Ko-Hsuan Hung, and the EMSA for the DmSNAP43 truncation construct was performed by Phuc Phan. All other data in this chapter were produced by this dissertation author.

CONCLUDING REMARKS

63 64

TBP, not TRF1, is preferentially utilized at U6 gene promoters

From the work of others [Takada et al., 2000], it was believed that transcription from all classes of Pol III promoters in D. melanogaster required TRF1 rather than TBP itself. As expected, ChIP experiments in Chapter 1 indicated that

TRF1 was indeed highly enriched at the promoters of tRNA genes in S2 cells. But in contrast, our data revealed that TBP was significantly more enriched than TRF1 at the

U6 promoters. These latter findings were consistent with the failure to detect TRF1 at

U6 promoters in genome-wide ChIP-on-chip experiments performed by Isogai et al.

[Isogai et al., 2007]. Furthermore, significant inhibition of U6 transcription in vitro was observed when TBP (but not TRF1) was immunodepleted from a nuclear extract.

In contrast, the opposite was true in the case of tRNA transcription, confirming that tRNA transcription required TRF1 but not TBP. Analogous experiments that employed RNAi to knock down TBP or TRF1 in cells led to the same conclusions regarding the preferential utilization of TBP by U6 genes and TRF1 by tRNA genes.

Our results do not agree with the conclusion by others that TRF1 is required for U6 transcription in Drosophila [Takada et al., 2000]. We believe that there is a simple explanation for this discrepancy. The primer extension assay used in our in vitro transcription experiments was designed to detect synthesis of only correctly initiated U6 snRNA transcripts. In the in vitro transcription assays of Takada et al.

[Takada et al., 2000], the transcription product was detected by labeling the synthesized RNA with [α-32P] GTP. Thus, not only U6 transcripts but also any RNA of discrete size transcribed from the plasmid template would have been detected. In

65 fact, the genomic DNA cloned into plasmid pDU6-1 used as a template by Takada et al. [Takada et al., 2000] contained a known aspartic acid tRNA gene (FlyBase annotation ID tRNA:D:96A) that is less than 1,000 bp from the U6:96Aa gene in the fly genome [Das et al., 1987]. Furthermore, we estimated that the tRNA gene utilized in our transcription assays [Verma et al., 2013] was transcribed roughly 100-fold more efficiently in vitro than the U6 gene, based upon the relative amount of primer radioactivity that was added to give comparable U6 and tRNA signals. Thus, the transcript labeled U6 in Fig. 1 of Takada et al. [Takada et al., 2000] is undoubtedly a misidentified tRNA transcript.

Since the ChIP assays detected slightly above background levels of TRF1 at

U6 promoters, the possibility of TRF1 functioning occasionally or under certain conditions to support U6 transcription cannot be ruled out. Also, the overexpression of

TRF1 caused an increase in the TRF1 occupancy measured at U6 promoters in cells.

Moreover, TRF1 (when added in amounts far greater than present originally in soluble nuclear fraction [SNF]) could restore U6 transcription in nuclear extracts depleted of both TBP and TRF1. Thus, U6 genes may be capable of utilizing TRF1 for their transcription on occasion or in the absence of TBP. However, the fact that U6 expression was sensitive to a reduction of TBP levels but not TRF1 levels both in vitro and in vivo provided strong evidence that TBP is utilized for the vast majority of U6 transcription in fruit flies.

A number of studies have revealed that transcription by Pol II of different populations of mRNA promoters differentially depends upon TBP or a TBP-related factor [Hernandez et al., 2007; Takada et al., 2000; Isogai et al., 2007; Hampsey,

1998; Hernandez, 1993; Crowley et al., 1993]. To our knowledge, the finding that different classes of Pol III promoters can likewise differentially utilize TBP or a TBP- related factor was not previously encountered. Most likely, the differential utilization of TBP and TRF1 at U6 and tRNA promoters arises from different pathways of preinitiation complex assembly (Fig. 1). In the case of tRNA genes, TFIIIC is required as an initial DNA-binding factor that recognizes the internal promoter. In contrast,

SNAPc is required to recognize the external promoter of U6 snRNA genes. It is possible that the utilization of TBP for U6 snRNA transcription in flies facilitates U6 coordinate regulation with that of the Pol II-transcribed spliceosomal snRNA genes that utilize TBP. The study in Chapter 1 was important not only because it corrected a long-standing error in the literature, but also because it demonstrated for the first time that Pol III can differentially utilize TBP and a TBP-related factor for transcription at different classes of Pol III promoters.

Figure 1: Schematic representation of transcription requirements at different classes of RNA polymerase III promoters. Fly tRNA genes utilize TFIIIC and TRF1 for their transcription by Pol III (top); in contrast, U6 genes require DmSNAPc and TBP to initiate Pol III transcription (bottom). Brf1 and Bdp1 are also shown in the diagrams.

Bdp1 acts as a bridge between DmSNAPc and TBP on the U6 snRNA gene promoter

It has been shown that TFIIIB is recruited to the promoters of tRNA genes by direct interactions with TFIIIC [Kassavetis et al., 1990]. The next question that arises here is how TFIIIB is recruited by DmSNAPc to form a stable pre-initiation complex at the U6 gene promoter. Work described in Chapter 2 investigated whether interactions between DmSNAPc and TFIIIB could be identified on the U6 promoter in vitro. Earlier work by others in the human system has shown cooperative binding between SNAPc and TBP thereby stabilizing TBP binding to the U6 TATA box

[Hinkley et al., 2003; Ma and Hernandez, 2002; Mittal and Hernandez, 1997; Saxena et al., 2005]. The results shown in chapter 2, likewise, revealed similar weak binding between DmSNAPc and TBP on the U6 promoter.

Surprisingly, however, a novel interaction between DmSNAPc and Bdp1 was detected: It was discovered that DmSNAPc could recruit Bdp1 to the U6 promoter, which is particularly significant as Bdp1 does not possess any DNA binding activity of its own. Analysis utilizing a series of N-terminal and C-terminal truncation constructs of Bdp1 localized a region of Bdp1 just C-terminal of the conserved SANT domain that was responsible for the interaction with DmSNAPc.

Furthermore, we found that the DmSNAPc-Bdp1 complex was able to recruit

TBP to the TATA box with much greater efficiency than DmSNAPc alone. Thus,

Bdp1 apparently acts as a bridge between DmSNAPc and TBP to establish a quaternary complex of the three proteins tightly bound to the U6 promoter DNA.

Interestingly, the same region of Bdp1 that is responsible for interaction with

DmSNAPc was found to be likely involved also in TBP recruitment to the U6 promoter.

In our view, the most intriguing finding of Chapter 2 is that the presence of a

U6 PSEA is crucial for the recruitment of Bdp1 by DmSNAPc. A 5-base-pair change that switched the U6 PSEA of the U6 promoter to a U1 PSEA abolished the ability of efficiently bound DmSNAPc to recruit Bdp1. This suggests that a conformation attained by DmSNAPc, when bound to a U1 PSEA, makes it incompatible for interaction with Bdp1. This finding provides new evidence in support of a model of snRNA gene RNA polymerase specificity previously proposed by our lab [Hung and

Stumph, 2011; Kang et al., 2014; Kim et al., 2010a, b; Li et al., 2004; Wang and

Stumph, 1998]. In that model, the specific DNA sequence of the PSEA acts as an allosteric effector of DmSNAPc conformation that restricts its ability to recruit only

Pol III GTFs to the U6 gene and only Pol II GTFs to the U1 gene. An updated model that incorporates these new findings is also presented in Chapter 2 that illustrates biochemical mechanisms behind RNA polymerase specificity on U1 v/s U6 promoter

DNA. Altogether, our results indicate that a network of very specific molecular interactions must occur to cooperatively establish a stable protein-DNA complex on the U6 promoter. This network must involve at least five conserved components in flies: a Pol III-specific PSEA, DmSNAPc (while bound to a Pol III-specific PSEA),

Bdp1, TBP, and the TATA box.

Because the study in Chapter 3 was unable to identify any region of

DmSNAP43 that is required for Bdp1 recruitment, additional experiments will be required in order to localize regions of DmSNAPc that are involved in interaction with

Bdp1. Experiments recently completed with two available DmSNAP190 truncations

(one N-terminal and one C-terminal) indicate that these two truncations more than likely do not interfere with Bdp1 recruitment.

The use of DmSNAPc truncations is complicated by the fact that very little if any of the three DmSNAPc subunits can be deleted without losing the DNA-binding activity of the complex [Hung et al., 2009]. Furthermore, it is quite possible that multiple regions of DmSNAP43 and/or of the other two subunits may be sufficient for

Bdp1 recruitment when examined by EMSA. Further experiments of this type would be fairly costly and may not meet with success.

In recent years, an alternative and superior method has become available for localizing regions of protein-protein proximity within stable protein complexes: cross- linking mass spectrometry. In this method, a chemical cross-linker is utilized to cross- link specific nearby amino acids (for example, the bifunctional cross-linker EMS cross-links lysines) within the 3-dimensional protein complex. Following protease digestion, peptides that are near to each other in the complex can be identified by mass spectrometry. An advantage of this method is that the wild type proteins can be used to form the complexes and subsequently analyzed. In this way, it should be possible to identify regions of DmSNAPc and of Bdp1 (and of TBP) that are in close proximity to each other while bound to U6 promoter DNA. A collaboration has been initiated to undertake these studies.

REFERENCES

Crowley, T.E., Hoey, T., Liu, J.K., Jan, L.Y., Tjian, R. (1993). A new factor related to TATA-binding protein has highly restricted expression patterns in Drosophila. Nature 361, pp. 557–561.

Das, G., Henning, D., Reddy, R. (1987). Structure, organization, and transcription of Drosophila U6 small nuclear RNA genes. J Biol Chem 262: 1187-93

Hampsey, M. (1998). Molecular genetics of the RNA polymerase II general transcriptional machinery. Microbiol. Mol. Biol. Rev. 62, 465–503.

Hernandez, N. (1993). TBP, a universal eukaryotic transcription factor?. Genes Dev. 7, 1291–1308.

Hung, K.H., and Stumph, W.E. (2011). Regulation of snRNA gene expression by the Drosophila melanogaster small nuclear RNA activating protein complex (DmSNAPc). Crit Rev Biochem Mol Biol 46, 11-26.

Hung, K. H., Titus, M., Chiang, S. C., and Stumph, W. E. (2009). A map of Drosophila melanogaster small nuclear RNA-activating protein complex (DmSNAPc) domains involved in subunit assembly and DNA binding. J. Biol. Chem. 284, 22568- 22579.

Isogai, Y., Takada, S., Tjian, R., and Keles, S. (2007). Novel TRF1/BRF target genes revealed by genome-wide analysis of Drosophila Pol III transcription. The EMBO Journal 26: 76-89.

Kassavetis, G.A., Braun, B.R., Nguyen, L.H., Geiduschek, E.P. (1990) S. cerevisiae TFIIIB is the transcription initiation factor proper of RNA polymerase III, while TFIIIA and TFIIIC are assembly factors. Cell 60, 235-245

Kim, M.K., Kang, Y.S., Lai, H.T., Barakat, N.H., Magante, D., and Stumph, W.E. (2010a). Identification of SNAPc subunit domains that interact with specific nucleotide positions in the U1 and U6 gene promoters. Mol Cell Biol 30, 2411-2423. Kim, M.K., Kang, Y.S., Lai, H.T., Barakat, N.H., Magante, D., and Stumph, W.E. (2010b). Identification of SNAPc subunit domains that interact with specific nucleotide positions in the U1 and U6 gene promoters. Mol Cell Biol 30, 5257.

Ma, B.C., and Hernandez, N. (2002). Redundant cooperative interactions for assembly of a human U6 transcription initiation complex. Mol Cell Biol 22, 8067-8078.

Mittal, V., and Hernandez, N. (1997). Role for the amino-terminal region of human TBP in U6 snRNA transcription. Science 275, 1136-1140.

Takada, S., Lis, J.T., Zhou, S., Tjian, R. (2000). A TRF1: BRF complex directs Drosophila RNA polymerase III transcription. Cell 101: 459-69.

Verma, N., Hung, K. H., Kang, J. J., Barakat, N. H., Stumph, W. E. (2013). Differential utilization of TATA box-binding protein (TBP) and TBP-related factor 1 (TRF1) at different classes of RNA polymerase III promoters. J. Biol. Chem. 288.38, 27564-27570

APPENDIX

A. Detailed protocol for chromatin immunoprecipitation assay (ChIP) B. Detailed protocol for in vitro transcription assay C. Detailed protocol for immuno-depletion assay D. Detailed protocol for nickel-chelate chromatography E. Detailed protocol for electrophoretic mobility shift assay (EMSA)

72 73

Appendix A. Detailed Protocol for Chromatin immunoprecipitation Assay (ChIP) I. Solutions and materials

Formaldehyde cross-linking solution original solution final solution Chemical conc. unit add unit conc. unit Tris-HCl (pH8.0) 1.0 M 1.400 ml 50.0 mM EDTA (pH8.0) 0.5 M 0.056 ml 1.0 mM EGTA 0.1 M 0.140 ml 0.5 mM NaCl 5.0 M 0.560 ml 100.0 mM Formaldehyde 37.0 % 0.757 ml 1.0 % final solution volume 28.0 ml * final volume is from all chemicals+ 25ml S2 cell culture. Note: Add each individual chemical directly to the 25 ml S2 cells in the order shown.

2M glycine Dissolve 7.5g glycine in 50 ml sterile d.d. water. Store at 4°C.

Sonication buffer original solution final solution Chemical conc. unit add unit conc. Unit Tris-HCl (pH8.0) 1.0 M 0.50 ml 10.0 mM EDTA (pH8.0) 0.5 M 0.10 ml 1.0 mM EGTA 0.1 M 0.25 ml 0.5 mM PMSF 0.2 M 0.125 ml 0.5 mM (add d.d. water to bring to the final final solution volume 50.0 ml volume. Store at 4°C.) Notes: PMSF is inactivated in aqueous solutions, so stock solution should be made in ethanol or isopropanol and only added to aqueous solutions immediately before use. Add 2.5 µl 0.2M PMSF per 1ml sonication buffer. Also, add 10 µl protease inhibitor cocktail (SIGMA) per 1 ml sonication buffer right before use.

Buffer for preparation of dialysis tubing Buffer I original solution final solution

Chemical conc. unit add unit conc. Unit sodium bicarbonate (NaHCO3) (powder) 100.0 % 20.0 g 2.0 % EDTA (pH 8.0) 0.5 M 2.0 ml 1.0 mM (add d.d. water to bring to the final final solution volume 1000.0 ml volume.)

Buffer II: 1mM EDTA (pH 8.0) 2ml 0.5M EDTA (pH 8.0)/1000 ml d.d. water.

6M Urea Dissolve 18.02 g Urea in 30 ml d.d. water. Adjust the volume to 50 ml with d.d. water. Store at room temperature. Use within 1-2 weeks.

ChIP buffer original solution final solution chemical conc. unit add unit conc. unit Tris-HCl (pH8.0) 1.0 M 10.0 ml 10.0 mM EDTA (pH 8.0) 0.5 M 2.0 ml 1.0 mM EGTA 0.1 M 5.0 ml 0.5 mM PMSF 0.2 M 2.5 ml 0.5 mM glycerol 100.0 % 100.0 ml 10.0 % Triton-X100 100.0 % 10.0 ml 1.0 % sodium deoxycholate (powder) 100.0 % 1.0 g 0.1 % (add water to bring to the final final solution volume 1000.0 ml volume. Store at 4°C.)

Notes: PMSF is inactivated in aqueous solutions, so stock solution should be made in ethanol or isopropanol and only add to aqueous solutions immediately before use.

Usually only 200 ml ChIP buffer is needed per dialysis, so add 500 µl 0.2M PMSF for 200 ml ChIP buffer.

Appropriate antiserum and pre-immune serum Anti-DmSNAP43 Ab (DmSNAP43 (03978) antibody 12-6-04 Nermeen) and Anti- FLAG polyclonal Ab (SIGMA, product code: F7425) are used in this case.

Immobilized Protein A sepharose (PIERCE, product code: 20333)

Note: Binding specificities and affinities of different antibody-binding proteins (protein A, G, A/G, and L) differ between source species and antibody subclass. In this case, protein A is selected because of its high affinity to rabbit IgG (anti- DmSNAP43 Ab and anti-FLAG polyclonal Ab). You may need to use other antibody- binding proteins if other antibodies are used in your application.

TE buffer Add 1 ml of 1M Tris-HCl pH 8.0 and 200 µl of 0.5 M EDTA pH 8.0 in a 100 ml cylinder. Fill d.d. water to 100 ml graduation. Filter to sterilize. Store at 4°C.

10 mg/ml BSA (NEB, product code: B9001S) Directly use the NEB 100X BSA (10 mg/ml) that comes with restriction enzymes.

Low-salt wash buffer original solution final solution chemical conc. unit add unit conc. unit Tris-HCl (pH8.1) 1.0 M 4.0 ml 20.0 mM EDTA (pH 8.0) 0.5 M 0.8 ml 2.0 mM NaCl 5.0 M 6.0 ml 150.0 mM SDS 10.0 % 2.0 ml 0.1 % Triton-X100 100.0 % 2.0 ml 1.0 % (add d.d. water to bring to the final final solution volume 200.0 ml volume. Store at 4°C.)

High-salt wash buffer original solution final solution Chemical conc. unit add unit conc. unit Tris-HCl (pH8.1) 1.0 M 4.0 ml 20.0 mM EDTA (pH 8.0) 0.5 M 0.8 ml 2.0 mM NaCl 5.0 M 20.0 ml 500.0 mM SDS 10.0 % 2.0 ml 0.1 % Triton-X100 100.0 % 2.0 ml 1.0 % (add d.d. water to bring to the final solution volume 200.0 ml final volume. Store at 4°C.)

Lithium wash buffer original solution final solution Chemical conc. unit add unit conc. unit Tris-HCl (pH8.1) 1.0 M 1.0 ml 10.0 mM EDTA (pH 8.0) 0.5 M 0.2 ml 1.0 mM LiCl 10.0 M 2.5 ml 250.0 mM NP-40 100.0 % 1.0 ml 1.0 % sodium deoxycholate (powder) 100.0 % 1.0 g 1.0 % (add d.d. water to bring to the final solution volume 100.0 ml final volume. Store at 4°C.)

ChIP elution buffer (freshly made) original solution final solution Chemical conc. unit add unit conc. unit sodium bicarbonate (NaHCO3) 1.0 M 10.0 ml 100.0 mM SDS 10.0 % 10.0 ml 1.0 % (add water to bring to the final final solution volume 100.0 ml volume. Do not chill.)

DPBS (Invitrogen, product code 14190) (Dulbecco’s PBS) Note: You can use any other 1X PBS from other vendors.

Protease Inhibitor Cocktail (SIGMA, product code P8340)

Sonicator (Branson sonifier 250 Analog, with microtip) (in Huxford lab)

Spectra/Por 2 dialysis tubing (MWCO 12-14000 Da, nominal flat width 10 mm, 0.32 ml/cm) (SpectrumLab, product code 132676)

Dialysis tubing clamps

Tris-HCl (pH6.5)

Proteinase K (2mg/ml)

QIAquick PCR purification kit (QIAGEN, product code 28104)

Platinum PCR SuperMix (Invitrogen, product code: 11306-016)

Appropriate forward and reverse primers for PCR reactions (200 ng/reaction)

U1Forward (5’-GTGTGGCATACTTATAGGGGTGCT-3’) and U1Backward (5’- GCTTTTCGATGCTCGGCAGCAG-3’) primers that amplify the promoter region of the U1:95Ca gene from -1 to -107 relative to the transcription start site are used in this case.

PCR machine (BioRad iCycler)

10X TBE

10X ChIP loading dye Add 2.1 ml 1% bromophenol blue to 2.5 ml glycerol in a 15 ml Falcon tube. Add water to bring to 5 ml. Vortex to mix. Store at room temperature.

II. Preparation of dialysis tubing

1. Cut the dialysis tubing into 15 cm pieces (~2 ml capacity/piece). 2. Boil the tubing in 800 ml buffer I in a glass beaker for 10 minutes with stirring. 3. Rinse the tubing thoroughly in d.d. water. 4. Boil the tubing in 800 ml buffer II in a glass beaker for 10 minutes with stirring. 5. Allow the tubing to cool, and then store it in cold room overnight (cover the beaker with aluminum foil). Make sure the tubing is always submerged.

Note: From now on, always wear gloves to handle the tubing. 6. Before use, wash the tubing inside and out with d.d. water.

III. Formaldehyde cross-linking, sonication, and dialysis

Day 1

1. Grow 3 plates (Corning 100 x 20 mm tissue culture plate) of Drosophila S2 cells to 90 % confluency. 2. Harvest cells: a. To remove cells adhering to the dish, pipet the medium over the cells gently several times. b. Pool cells from all plates and transfer 25 ml of cells into a 50 ml Falcon tube. 3. Add chemicals of formaldehyde cross-linking solution into the 25 ml cells for cross-linking. Mix well. Incubate at room temperature for 10 minutes on a rotating wheel. 4. Add 3.8 ml 2M glycine to final 240 mM to quench the cross-linking reaction.

5. Spin the cells at 700 g, 4°C for 10 minutes (SORVALL, Legend RT). Discard the supernatant.

Note: The supernatant contains formaldehyde, which is a carcinogen. So it is important NOT to directly drain the supernatant into the sink or trash can. Instead, collect the supernatant into a 50 ml Falcon tube and toss it into the chemical hazard container. 6. Resuspend the cells with 10 ml ice-cold DPBS. Centrifuge at 700 g, 4°C for 10 minutes (SORVALL, Legend RT). Discard the supernatant. 7. Resuspend the cells with 1 ml sonication buffer with 2.5 µl 0.2M PMSF and 10 µl protease inhibitor cocktail. Transfer the suspension to a chilled 15 ml conical-bottom Falcon tube. 8. Sonicate the suspension on ice with the following condition: microtip, 60% duty cycle, 1.5 output, 30 second on/1 minute off, 10 cycles (14 minutes total). 9. Transfer sonicated solution to 2 chilled 1.5 ml screw-cap tubes (500 µl/tube). Centrifuge at 13,000 rpm, 4°C for 10 minutes (EPPENDORF, Centrifuge 5415D. In the cold room). 10. Pool supernatant from both tubes to a chilled 15 ml Falcon tube. Mix the solution with equal amount (~1ml) of 6M Urea.

Note: Mix well. Save a 50 µl aliquot separately in a 1.5 ml screw-cap tube at - 20°C in case it is necessary to determine DNA size. 11. Working in the cold room, take out the prepared dialysis tubing (Step II-6). Use a dialysis clamp to close one end of the tubing. Pipet the solution from Step III-10 into the tubing. Close the other end with another clamp. 12. Prepare a beaker containing 200 ml of ice-cold ChIP buffer (prepared the day before). Add 500 µl 0.2M PMSF per 200 ml ChIP buffer right before dialysis. Put tubing into the buffer and stir overnight in the cold room for dialysis.

IV. Preparation of 50% protein A sepharose

Day 2

1. Gently vortex to thoroughly suspend the immobilized protein A sepharose in the vial.

Note: Ratio of volume of suspension to packed gel is 2 to 1 in the vial.

2. Using a P-1000 with ~2 mm cut-off end of the tip, immediately transfer 200 µl suspended resin (100 µl packed resin) from the vial to a chilled 1.5 ml screw- cap tube.

Notes: Always use tips cut off at the end to handle the resin. More than 200 µl resin may need to be prepared depending upon the number of samples to be done.

3. Centrifuge at 2500 g, 4°C for 3 minutes (EPPENDORF, Centrifuge 5415D. In the cold room). Pipet off the supernatant. 4. Continue working with EPPENDORF Centrifuge 5415D. Wash resin twice with 1 ml sterile d.d. water. a. Resuspend beads with 1 ml sterile d.d. water. b. Centrifuge at 2500 g, 4°C for 3 minutes. c. Pipet off the supernatant. d. Repeat steps a-c. 5. Wash resin twice with 1 ml TE buffer. a. Resuspend beads with 1 ml TE buffer. b. Centrifuge at 2500 g, 4°C for 3 minute. c. Pipet off the supernatant. d. Repeat steps a-c. 6. Wash resin once with 1 ml TE+BSA (900 µl TE buffer +100 µl 10 mg/ml BSA). a. Resuspend beads with 1 ml TE+BSA. b. Centrifuge at 2500 g, 4°C for 3 minute. c. Pipet off the supernatant. 7. Resuspend beads with 100 µl TE+BSA (90 µl TE buffer +10 µl 10 mg/ml BSA). Now you have 200 µl of prepared 50% protein A resin. Store at 4°C.

Note: Among 200 µl prepared 50% resin, 80 µl is for pre-clearing, 35 µl is for each pre-immune serum and each anti-serum treated samples.

V. Pre-clearing and immunoprecipitation

1. Remove a clamp from one end of the dialysis tubing (Step III-12). Pipet out the solution into a chilled 1.5 ml conical screw-cap tube. Centrifuge at 13,000 rpm, 4°C for 10 minutes (EPPENDORF, Centrifuge 5415D. In the cold room). 2. Transfer the supernatant (~1 ml) into a chilled 1.5 ml screw-cap tube. This is the chromatin solution. 3. Add 80 µl of suspended protein A resin (Step IV-7) to the chromatin solution for pre-clearing. End-over-end rotate at 4°C for 30 minutes.

Note: This step (pre-clearing) is important to reduce the background signals. 4. Centrifuge at 13,000 rpm, 4°C for 5 minutes (EPPENDORF, Centrifuge 5415D. In the cold room). 5. Aliquot the supernatant into chilled 1.5 ml screw-cap tubes (200 µl for input; 150 µl for each pre-immune serum and each antiserum). Save the remainder in a chilled 1.5 ml screw-cap tube and store it along with the input tube at -80°C. Note: Input sample serves as positive PCR control. Pre-immune serum serves as negative immunoprecipitation control.

6. Add 4 µl of antiserum or pre-immune serum into each corresponding tube containing pre-cleared chromatin solution. End-over-end rotate at 4°C overnight.

Note: The amount of antiserum added may need to be optimized according to what antiserum you use. In this case, the antiserum used are anti-DmSNAP43 Ab and anti-FLAG polyclonal Ab (SIGMA, product code: F7425).

Day 3

7. Using a P-100 with the bottom of the tip cut off, add 35 µl prepared 50% protein A resin (Step IV-7) into each pre-immune tube and each antiserum tube. End-over-end rotate at 4°C for 2 hours. 8. Spin down the resin at 2500 g, 4°C for 3 minutes (EPPENDORF, Centrifuge 5415D. In the cold room). The resin is the important fraction, but save the supernatant (as “Flow through”) in a 1.5 ml screw-cap tube at -80°C in case it should be needed. 9. Continue working in the cold room and with EPPENDORF Centrifuge 5415D. Wash resin 3 times with 1 ml ice-cold low-salt wash buffer. a. Resuspend beads with 1 ml low-salt wash buffer. b. End-over-end rotate at 4°C for 5-10 minutes. c. Centrifuge at 2500 g, 4°C for 3 minute. d. Pipet off the supernatant. e. Repeat steps a-d for 5 more times. 10. Wash resin 3 times with 1 ml ice-cold high-salt wash buffer. a. Resuspend beads with 1 ml high-salt wash buffer. b. End-over-end rotate at 4°C for 5-10 minutes. c. Centrifuge at 2500 g, 4°C for 3 minute. d. Pipet off the supernatant. e. Repeat steps a-d for 2 more times. 11. Wash resin twice with 1 ml ice-cold lithium wash buffer. a. Resuspend beads with 1 ml lithium wash buffer. b. End-over-end rotate at 4°C for 2 hours. c. Centrifuge at 2500 g, 4°C for 3 minute. d. Pipet off the supernatant. e. Resuspend beads with 1 ml lithium wash buffer. f. End-over-end rotate at 4°C overnight (the overnight wash is believed to be important). g. Centrifuge at 2500 g, 4°C for 3 minute. h. Pipet off the supernatant.

Day 4

12. Wash resin with 1 ml ice-cold TE buffer. a. Resuspend beads with 1 ml TE buffer. b. End-over-end rotate at 4°C for 5 minutes. c. Centrifuge at 2500 g, 4°C for 3 minute. d. Pipet off the supernatant. 13. Resuspend resin with 1 ml TE buffer. Transfer the resin to another chilled 1.5 ml screw-cap tube to eliminate non-specific DNA bound on the tube wall. 14. Centrifuge at 2500 g, 4°C for 3 minute. Pipet off the supernatant.

VI. Elution, reverse cross-linking, and DNA purification

1. Add 250 µl of freshly made elution buffer to the resin (Step V-14) to elute the immunoprecipitated protein-DNA complexes. Vortex briefly to mix well. 2. End-over-end rotate at room temperature for 15 minutes. Centrifuge at 2500 g, room temperature for 3 minute (EPPENDORF, Centrifuge 5424). 3. Transfer the supernatant (eluate) to a 1.5 ml screw-cap tube. 4. Add another 250 µl of elution buffer to the resin to elute again. Repeat Step VI-2. Pool eluate from both elutions together in a 1.5 ml screw-cap tube (~500 µl total). Also, prepare input DNA by adding 300 ul elution buffer to 200 µl thawed input sample (Step V-5) to make final volume 500 µl. 5. Add 20 µl of 5M NaCl to eluate and input DNA. Mix well and incubate at 65°C for 4 hours to reverse crosslinks. 6. Add 10 µl of 0.5M EDTA pH8.0, 20 µl of 1M Tris-HCl pH6.5, and 10 µl of 2 mg/ml proteinase K. Mix well and incubate at 45°C for 1 hour to digest proteins. 7. Using QIAquick PCR purification kit (QIAGEN), follow the manufacturer’s instructions to purify the immunoprecipitated DNA and input DNA. Store the purified DNA at -20°C.

Notes: Use 2500 µl of PB buffer (as 5 volumes PB: 1 volume sample) in the first step (binding step); use 30 µl of TE buffer to elute the purified DNA in the last step. 30 µl of purified DNA is enough for 15 PCR reactions.

Now you will have at least 3 purified DNA sample: 1 for input DNA; 1 (or more) for pre-immune serum precipitated DNA; 1 (or more) for antiserum precipitated DNA.

VII. PCR reaction

Day 5

1. Prepare the PCR reaction by using purified pre-immune, anti-serum precipitated DNA, and input DNA (Step-VI-7) as instructed below:

Reagents amount (µl) Platinum PCR SuperMix 45 Forward primer (0.08 µg/µl) 2.5 Reverse primer (0.08 µg/µl) 2.5 DNA from ChIPs 2 Total 52

2. Use the following program to run the PCR reaction:

Cycle1: (1x) Step 1 94℃ 2:00 min hot-start Cycle2: (28x) Step 1 94℃ 0:30 min denature Step 2 61℃ 0:45 min anneal Step 3 72℃ 1:00 min extend Cycle3: (1x) Step 1 72℃ 10:00 min final extension Cycle4: (1x) Step 1 4℃ forever

Note: The PCR condition may need optimization. Change the anneal temperature according to the Tm of your primers. Change the extension time according to the length of your PCR products (1 min for 1 kb, but not less than 1min). 3. Prepare the DNA loading sample as instructed below by using PCR products amplified from pre-immune or antiserum-precipitated or input DNA. Also prepare the DNA marker.

reagents amount (µl) DNA 18 10X ChIP loading dye 2 Total 20

reagents amount (µl) DNA 4 10X ChIP loading dye 2 1X TBE 14 total 20

amount (µl) DNA 2 10X ChIP loading dye 2 1X TBE 16 Total 20

4. Run the prepared DNA samples on a 8% native polyacrylamide gel (with 20 wells) in 1X TBE at 180 V until bromophenol blue migrates to the bottom of the gel (~2 hr and 40 min).

Notes: make an 8% polyacrylamide gel as instructed below: Add 16 ml 30% acrylamide (30:0.8 acrylamide: bisacrylamide), 37.6 ml d.d. water, 6 ml 10X TBE, 420 µl 10% APS and 90 µl TEMED in a 125 ml flask. Swirl to mix. Immediately pour the solution in a pre-assembled gel apparatus. Insert the 20 well comb. Wait for 20 minutes to solidify.

Acrylamide is neurotoxic. Always wear protection while handling it. 5. Stain the gel with 0.5 µg/ml ethidium bromide (e.g. add 100 µl 5 mg/ml ethidium bromide in 1000 ml 1X TBE buffer) for 10 minutes. Destain the gel in deionized. water for 30 minutes. 6. Put the gel on a UV box. Turn on the UV and observe the DNA bands (wear protection). Take a photo of the gel for your record. Save the digital file.

VIII. qPCR reaction

7. Prepare the qPCR reaction by using purified pre-immune, anti-serum precipitated DNA, and input DNA (Step-VI-7) as instructed below: (reaction/well)

reagents amount (µl) 2X SYBR 5 Forward primer (10 µM) 0.3

Reverse primer (10 µM) 0.3 Nuclease free water 2.4 DNA from ChIPs (diluted) 2 Total 10

Notes: Since qPCR is highly sensitive, for quality control reasons, each sample should be run in triplicate. Input DNA serves as positive control; Extra reactions that use Nuclease free water instead of DNAfrom ChIPs should be included as negative no-template control.

Usually Input DNA is diluted 12X and other ChIPed DNA is diluted 3X to add into qPCR reaction.

Appendix B. Detailed Protocol for In Vitro Transcription Assay

I. Solutions and Materials Stock Solutions:

When making solutions, always wear gloves, and always use RNase-free H2O that you have personally ordered. All glassware (2-graduated cylinder, 2-10ml graduated cylinders, 2-100ml beakers, 3-50ml beakers, 3-50ml graduated cylinders, 1-brown screw capped 100ml glass bottle), metal spatulas, and stir bars should be autoclaved to destroy RNase.

1M MgCl2 Dissolve 203.3 g (2.033 g) of MgCl2.6H2O in 800 ml (8 ml) of ddH2O. Adjust the volume to 1 L (10 ml) with ddH2O. Dispense into aliquots in a brown screw capped bottle and sterlize by autoclaving.

1M MnCl2 Dissolve 197 g (1.97 g) of MnCl2.4H2O in 1 L (10 ml) ddH20. Autoclave. Store at room temperature.

+ 1 M Hepes, K , pH 7.6 (100 ml) Add 23.83 g of HEPES to about 60 ml of dH2O in a 150 ml beaker. Add a stir bar, place on a stir plate, and stir. Slowly add solid KOH until the pH is 7.6. Pour the solution into a 100 ml graduated cylinder and add dH2O to the 100 ml mark. Store in freezer.

HEMG, pH 7.6 (10 ml) Final concentration 8.6 ml dH2O (RNase-free) 250 ul 1 M HEPES, K+, pH 7.6 25 mM HEPES 125 ul 1 M MgCl2 12.5 mM MgCl2 2 ul 0.5 M EDTA, pH 8.0 0.1 mM EDTA 1 ml 100% Glycerol 10% Glycerol Store in freezer

Transcription Stop (50 ml) Final concentration 40.5 ml dH2O 2 ml 0.5 M EDTA, pH 8.0 20 mM EDTA 2.5 ml 4 M NaCl 200 mM NaCl 5 ml 10% (w/v) Sodium Dodecyl Sulfate 1% SDS Store at room temperature

Dithiothreitol (1 M; 1 ml) 154.3 mg DTT 1 ml dH2O Store in freezer

Sodium Acetate-NaOAc (3M; 50 ml) 24.4 g NaOAc Make up to 50 ml with dH2O Store in refrigerator

Sodium Acetate-NaOAc (0.3M; 50 ml) 5 ml 3 M NaOAc 45 ml dH2O Store in refrigerator

Ethanol, 100% (50 ml each) Uncap a fresh, unused 500ml bottle of ethanol and pour into ten 50ml conical polypropylene tubes. Store in freezer.

Sevag (Chloroform/Isoamyl Alcohol 19:1) Pipet 5 ml of Isoamyl Alcohol into a 100 ml brown screw-capped glass bottle. Add 95 ml of Chloroform into a 100 ml graduated cylinder, and pour into the brown glass bottle. Store at room temperature.

5x Annealing Buffer (10 ml) 6.75 ml dH20 (RNase-free) Final 5x Concentration 100 ul 1 M Tris-HCl, pH 7.8 10 mM Tris-HCl, pH 7.8 20 ul 0.5 M EDTA, pH 8.0 1 mM EDTA, pH 8.0 3.13 ml 4 M KCl 1.25 M KCl Store in freezer

10% Denaturing Acrylamide Add 47.5 g acrylamide [NOTE: Wear protective clothing/mask/gloves during the preparation], 2.5 g bis-crylamide, and 100ml 10X TBE buffer to 150ml ddH2O. Stir to dissolve. Slowly add 210 g urea. Moderately (DO NOT allow the temperature of the solution over 50 degrees else the acrylamide will evaporate) heat to facilitate the dissolving of the urea. After dissolving, the final volume should be around 500 ml. Add ddH2O to make 500 ml if necessary. Sterilize by passage through 0.22um filter. Store in refrigerator.

10X TBE Buffer Add 108 g Tris base, 55 g boric acid, and 40 ml 0.5M EDTA pH 8.0 to 800 ml distilled water. Stir to dissolve. Bring to 1 litre with distilled water. Store in refrigerator.

2x Denaturing Loading Buffer (10 ml) 10 ml 100% deionized Formamide 20 mg Xylene Cyanol 20 mg Bromophenol Blue Store in refrigerator

α-amanitin (50 ug/ml; 50 ul each)---highly toxic! Put used pipet tips into a biohazard waste container. Inside the hood, with a dust mask on, remove 30 ul of the concentrated 1 mg/ml α-amanitin stock solution and pipet into a pre-labeled tube. Return the concentrated stock to the freezer. Then pipet 2.5 ul of this smaller stock solution into 10 1.5 ml screw-capped tubes, each containing 47.5 ul dH2O (Rnase-free) for a final volume of 50 ul each. Store the labeled tubes (α- amanitin, 50 ug/ml) in the freezer.

Glycogen (1 ml) Add 20 mg powdered glycogen to 1 ml dH2O Store in freezer

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Labile solutions (make fresh every two months):

Ribonucleotide Triphosphates (5 mM each; 200 ul) 100 mM rNTPs are purchased from Boehringer Mannheim 10 ul each of 100 mM rATP, rCTP, rGTP, rTTP 160 ul of dH2O (RNase-free) Store in freezer

Extension Mix (5.4 ml; 140 reactions) ---toxic! Put all waste (needles, tubes, pipet tips, etc.) into a biohazard waste container.

First, with a 16 gauge needle, inject 1 ml of 100% Ethanol into the 2 mg bottle of Actinomycin D (highly toxic, INHIBITS TRANSCRIPTION; NOTE: do not remove the steel cover on the cap of the bottle) inside the hood. Mix by swirling the bottle side to side. With another 16 gauge needle, remove about 400 ul (0.4 ml) of the yellow Actinomycin D from the bottle, and "inject" carefully into a 1.5 ml screw-capped tube. Then pipet 62.5 ul of Actinomycin D (Out of the 0.4 ml screw capped tube) each into six 1.5 ml screw-capped tubes, labeled Extension Mix. If you have removed the steel cover of the actinomycin D bottle, then transfer rest of the actnimycin D solution of 0.4 ml

tube into another screw capped tube along with the the remaining solution in the bottle, cover it with aluminium foil and put it at -20°C. Put the caps on the tubes, transfer them to the speedvac, remove the caps, and spin the tubes to dryness (45 min). During this time, prepare the rest of the extension mix ingredients in six different screw-capped tubes than the ones spinning:

Important: add components in the following order

777 ul dH2O (RNase-free) 62.5 ul 1 M Tris-HCl, pH 8.3 12.5 ul 100 mM MnCl2 Keep on ice

Then add 25 ul each of dATP, dCTP, dGTP, and dTTP (100 mM each, Boehringer Mannheim) into a new screw-capped tube, and dilute with 150 ul RNase-free dH2O.

Add 35.6 ul of this dNTP solution into each of the six tubes already containing Tris and MnCl2.Vortex briefly. Then add 12.5 ul 1 M Dithiothreitol (DTT) to each tube. Vortex again.

After the tubes containing Actinomycin D have evaporated to dryness, put the caps on the tubes and transfer them to the hood. With a P1000, pipet the contents of the six tubes (Tris, MnCl2, dNTP's, DTT) into the Actinomycin D- containing tubes. Vortex vigorously for at least 1 minute, or until the orange pellet at the bottom is gone (can take up to 10 min to dissolve the orange pellet). Store in freezer, wrapped in aluminum foil (Actinomycin D is light- sensitive). If you do not expect to be doing a lot of transcription reactions, it may be better to prepare fewer than 6 tubes. Day before the experiment: be sure to write out the templates to be used and any other variables in the experiment in your notebook. Also read the MSDS for α-amanitin, Actinomycin D, Phenol, Chloroform, and Acrylamide. Always wear gloves for protection and to avoid RNase contamination.

II. Transcription reactions

1. Get two buckets of ice. Fill each to the top. Check to make sure that the two water baths are at 37°C and 45°C. Remove polyacrylamide from the refrigerator. Check the oil in the speed vacuum pump. 2. Thaw the following solutions (taken from the -20oC freezer) by rolling between one's fingers. 1 M Dithiothreitol (DTT) 1 M HEPES, K+, pH 7.6

5 mM rNTP solution (pre-mixed, contains 5 mM ATP, GTP, CTP, UTP) 0 M HEMG* Each DNA template, 0.2 ug/ul (prepared using Qiagen maxi-preps). Each should have been previously sequenced and also examined for integrity by agarose gel electrophoresis. NOTE: Also, 99% of this 0.4 ug of the U1 and U6 never get transcribed to RNA, may be because of some competition going on in between Histones and GTFs.

CALCULATION to make 50 ul of each DNA template (0.2 ug/ul): Concentration of the template * Amount required (X) / 50 ul = 0.2 ug/ul For U1 WTA DNA Template: 2.55 ug/ul * X / 50 ul = 0.2 ug/ul X = 3.92 ul + 46.08 ul RNase-free water For U6 WTD DNA Template: 2.56 ug/ul * X / 50 ul = 0.2 ug/ul X = 3.91 ul + 46.08 ul RNase-free water For tRNA DNATemplate: 2.785 ug/ul * X / 50 ul = 0.2 ug/ul X = 3.6 ul + 46.4 ul RNase-free water *The "zero M" means no KCl has been added to this buffer. Different concentrations of KCl may be used according to the type of promoter being used. 3. When the above solutions are thawed, vortex them for 5s. Spin tubes for 3s. Place all tubes on ice. 4. Go to the enzyme freezer with an ice bucket and obtain a tube of RNasin (Promega, 40 U/ul). Put in the ice bucket and return to bench. 5. Place one 1.5 ml screw-capped eppendorf tube on ice. Using only autoclaved (unbroken seal) pipet tips, prepare a transcription pre-mix, which contains components common to all reactions. If you are going to do 8 transcription reactions, prepare enough pre-mix for 10 reactions as follows:

Chemical For Each Rxn For 10 Rxns

rNTPs 2.5 ul 25 ul

HEPES 0.5 ul 5 ul

0M HEMG 0.5 ul 5 ul

1M DTT 0.1 ul 1 ul

RNasin 0.7 ul 7 ul

Final Volume 4.3 ul 43 ul

6. Again, using Fisher screw-capped eppendorf tubes, place the appropriate number of reaction tubes into the rack, and number them 1, 2, 3, etc. 7. Fill a bucket with tap water, put in a thermometer, and adjust temperature of o H2O to 25 C (adjust with either ice or warm water taken from a water bath). 8. Pipet the required amount of RNase-free H20 into each of the tubes as required (check experimental plan table).

Uninhibited Tubes w/ α- Tubes w/ Tubes w/ Both tubes amanitin Tagetitoxin inhibitors

1.7 ul 0 ul 0 ul 0 ul

9. Pipet 4.3 ul of the reaction mixture made in step 5 into each tube. 10. Pipet 2.0 ul of diluted DNA (0.2 ug/ul; 0.4 ug/ 400ng) of each template (U1 and U6) into each appropriately labeled tube. Put all buffer and DNA tubes back in the freezer. 11. Take the 20 U/ul tube of tagetitoxin out of the refrigerator. Vortex 5s, spin 3s. Pipet 2 ul of tagetitoxin (40 U) into the appropriate tubes (check notebook). Put the tagetitoxin back in the refrigerator. 12. Take out a 50 ug/ml stock of Rnase free α-amanitin from the freezer. Thaw the α-amanitin by itself in the hood. Pipet 2 ul of the α-amanitin into each appropriate tube in the hood. Bring the rack of tubes back to the bench. Return the α-amanitin stock to the freezer. 13. Remove one of the appropriate tubes of SNF from the liquid nitrogen tank (after experimentally choosing an SNF date for further work, they should be aliquotted into screw-capped tubes of 200 ul each). Thaw the tube by hand, and then place on ice. Mix the thawed SNF thoroughly by finger flicking. Spin 3s. Pipet 15 ul of the SNF into each reaction tube. Before changing tips, mix the SNF with the rest of the tube's contents by pipetting 3 times. [NOTE: Put the box with the remaining SNF tubes at -20 until you use the desired ones and then put all of them back in the liquid nitrogen together.] 14. Close the tubes, put the SNF back in the liquid nitrogen (if there is a sufficient volume). Further mix the tubes by finger flicking. Spin 3s.

II. Termination of Transcription Reactions

15. Place the tubes in a yellow 16-tube holder, and place the holder in the 25oC H2O bucket. Incubate tubes for 60 min. 16. During this time, thaw the labeled recovery standard and glycogen (20 ug/ml) solutions in the 37oC water bath. Vortex 5s, spin 3s. 17. Weigh out 1.5-3.0 mg of proteinase K and place in an empty tube. Add an appropriate amount of RNase-free H2O such that the final concentration of proteinase K is 12.5 mg/ml (ex. 2.5 mg would have 200 ul H2O added). 18. Add the following volume of solutions to an appropriate tube, depending upon the total number of transcription reactions being done: Add Transcription Stop, then Glycogen [It is a polysaccharide which is used as a carrier and helps DNA and RNA precipitation during ethanol precipitation], vortex, then add Proteinase K and Recovery Standard, then mix by finger flicking.

Total # Rxns Transcription Glycogen Proteinase K Recovery Stop Standard 2-4 500 ul 6.25 ul 10 ul 1500 cpm eq. 4-8 1000 ul 12.50 ul 20 ul 3000 cpm eq. 9-12 1500 ul 18.75 ul 30 ul 4500 cpm eq. 13-16 2000 ul 25.00 ul 40 ul 6000 cpm eq.

NOTE: 1. Recovery standard is a synthetic oligonucleotide, which is radio-labeled with p32. It shows you how much have you recovered from the in-vitro transcription reaction. It serves as a positive control and a band of recover standard should be seen in each lane on the film along with the respective transcripts. a. If a Recover standard band appears but there is no band for the transcripts, this indicates that the transcription did not take place. b. If there is no Recovery standard band on the film, this indicates that you have lost the samples. 2. CPM is not same as the number of molecules of identical oligonucleotide but it is the measurement of the number of beta particles emitted by the radiolabeled oligos. * The counts per minute (cpm) of recovery standard per reaction tube are 300 cpm in this example. This number will vary according to the length of time of exposure for each experiment. Note: Test a 105ul sample of this transcription stop mix in the scintillation counter. Prepare ethanol/dry ice bath. 19. Prepare for phenol / choloroform extraction [This is done to extract all the proteins such as RNA Pol, DmSNAPc, GTFs etc in the organic phase] and

ethanol precipitation of the transcription reactions: place two sets of screw- capped tubes in new rack(s), and number them accordingly, two at a time (ex. 1, 1, 2, 2, 3, 3, etc.). The racks have 5 holes vertically; put these tubes in the 3rd and 5th holes. Skip a hole horizontally when placing the empty tubes. Unscrew the tubes, but leave the caps on. 20. Take the rack of tubes to the hood together with phenol, chloroform, and a 12 ml round bottom polypropylene 2059 tube (in a styrofoam holder). Into the 12 ml tube pipet 250 ul chloroform and 250 ul phenol per transcription reaction (i.e., for eight reactions, pipet 2 ml phenol and 2 ml chloroform). Put the cap on tightly and vortex to mix. 21. After the 60 min. transcription incubation, remove the tubes from the 25oC water bath and place them in order into the first row of holes in the rack(s). Using one tip, pipet 105 ul of the transcription stop mix into each tube (do not touch the tubes with the pipet tip). Close the tubes, mix by gentle finger flicking and wait for 5-10 min, keeping the tubes at room temp. 22. During this time, pipet 400 ul of chloroform (not phenol/chloroform) into the middle set of tubes in the rack. The samples are to be extracted once with phenol/chloroform and once with chloroform. 23. After the proteinase K digestion (step 21) has gone to completion (5-10 min.), pipet 200 ul of 0.3M NaOAc into each tube (same pipet tip), and then pipet 400 ul of the phenol / chloroform mix into (up to) 8 reaction tubes (same pipet tips), or one rack of tubes. 24. Screw these tubes shut, place them in a completely empty rack, and put them on your bench. Vortex each tube (up to five tubes at a time) for 30s, then spin all tubes for 3-6 min. (<9 tubes = 3 min.; >8 tubes = up to 6 min.). During this time pipet 400 ul of the phenol / chloroform mix into any remaining tubes in the hood, vortex 30 s. At the beginning of the last vortex, stop the microfuge. The microfuge should take at least 30s to stop, allowing for a full 30s vortex of the last set of tubes. 25. Take the newly spun tubes out of the microfuge, and (if more than 8 total tubes) put in the remaining tubes. Spin those 6 min. 26. Take the newly spun tubes into the hood, place back into the rack in their 1st row position, and loosen their caps. Open the α-amanitin / actinomycin D waste container. Set the P200 pipet to "200". 27. Take the first tube's cap off (i.e., tube #1), and lay it down close to the tube. With a P200 pipet, extract the top (aqueous) layer out of the tube and into the middle row #1 tube (that already contains chloroform). It will take 2 pipettings to remove the 332 ul of upper aqueous layer. Tilt the tube 45oC, so that the aqueous layer facing away from you rolls around to face you (pipet out that, too). Expel used pipet tips, tubes, and caps in the waste container. 28. Put the organic phase tube and its cap into the waste container, and repeat step 27 for all of the tubes in the rack. 29. Screw the caps on to the middle set of tubes, put them in the empty rack, and put them back on your bench.

30. Vortex 30sand spin 3 min, and make sure the microfuge is spinning until the 2nd vortex, then press stop. Vortex the final set of tubes, then replace them with the tubes in the microfuge. If you have 8 or fewer tubes, this is the final spin before the ethanol precipitation. If you have 9 or more tubes, repeat steps 27-30 for the second set of tubes. 31. Remove the upper aqueous phase (about 332 ul) from the choloform phase in each tube, pipetting into the appropriately labeled empty tubes in the 5th row of holes in the rack. Be careful not to pipet any of the chloroform phase in with the sample. Close the waste container. Put the rack of tubes on your bench. 32. With one pipet tip, add 1 ml of cold 100% ethanol from the freezer to each tube. Close caps and vortex (up to 5 at a time) for 30s. Incubate samples in ethanol/dry ice bath for 15 min. Spin in cold room for 30 min. The numbers on the tubes should be facing out in order to locate the pellet when the spin is done.

IV. Annealing the Primer and Primer Extension Reactions

33. Put on a new pair of gloves, safety goggles, a lab coat, and radioactive body detector and finger ring. With a plastic shield and beta-block (4-hole plastic bar), take out the radiolabeled primer and 5x annealing buffer. Thaw them in the 37oC water bath. Remove promptly upon thawing. 34. Calculate the volume of primer needed to give each reaction about 600,000 cpm of primer (ex. 8 reaction tubes = 4,800,000 cpm). Perform all subsequent steps behind a plastic shield, preferably the large plastic bench shield. When the primer and annealing buffer have thawed, vortex each 5s and spin 3s. Add the appropriate amounts of primer and annealing buffer to give about 12 ul of volume (600,000 cpm / reaction tube). For example, if you calculated 15 ul of primer (at 335,000 cpm/ul) to add for 8 reactions, then add 20 ul 5X annealing buffer and 65 ul H2O for a final volume of 100 ul. Put the primer and annealing buffer tubes back in the freezer (put the primer in a large beta-block tube holder). NOTE: The primers used will be: 1. 1211Z Primer: This primer anneals to the plasmid vector pUC 118 and therefore works for both U1 and U6 genes thereby giving different size of the transcription products. 2. tRNA Maxi Primer: This primer is used for the reverse transcription of the tRNA. 35. Put 400 ml of dH2O into a 2L beaker, put the beaker on a heat/stir plate, and turn on the heat to about 2/3 maximum. Put a thermometer in the beaker. 36. Put 10 ul of the newly mixed primer/annealing buffer into a non screw-capped eppendorf tube, and count in the scintillation counter. If >600,000 cpm or <400,000 cpm, then adjust accordingly.

37. When the 30 min. spin in the microfuge is over immediately take the tubes back to your bench and pipet off about 2/3 of the ethanol with your p-1000 set at 1000 and then with a P200 pipet set at 200, carefully take the rest of the liquid out from the other side of the pellet (with the pipet tip on the bottom). Always check to see if the pellet is there afterwards, and check each tip for signs of a pellet. Leave the caps off as you go. 38. When you have removed all of the ethanol you can from the last tube, go back and starting with the first tube carefully pipet out the rest of the liquid in each tube. Avoid pellet! 39. With one pipet tip, place 10ul of primer/annealing buffer onto the middle side of each tube and screw the caps on. Tap the tubes so the solution falls to the bottom. Discard the tip and any remaining primer solution in the radioactive trash. 40. With your face and body shielded by the large bench shield, vortex the tubes (3-5 at a time) for 20s. Spin 3s. 41. While the tubes are spinning down, check the temperature of the water. Adjust the heat control so that the water will be between 70-80oC (you may have to take the beaker off the heat plate if over 85oC). 42. Put the newly spun tubes in the yellow tube holder and put the holder in the water bath for 2 min at 70-80oC. Then put the holder in the 45oC water bath for 60 min, and put the plastic shield on top of the bath to let others know there is radioactivity in the bath.

III. Assembling the Gel Apparatus

43. If running the gel today, while the tubes are incubating in the 45°C water bath, assemble the gel: Wearing gloves, get two large plates (long and short, both thick glass). Take one at a time to the sink and scrub thoroughly with dish soap and a paper towel. Rinse thoroughly. Take two thin spacers and wash them thoroughly with dish soap and water, and rinse thoroughly. Wipe the water off the plates with Kimwipes. Then squirt room temperature 100% ethanol on both plates, and wipe with Kimwipes. Squirt ethanol on the sides of the both plates that will be inside facing the gel, and scrape it off with a razor blade Then squirt about 2 square inches of Gelslick on the middle of the small plate. Spread the Gelslick over all of the small plate with Kimwipes. After spreading Gelslick on the small plate, spread residual Gelslick from the Kimwipe on the large plate. Wait about 5 min. 44. After the five minutes, squirt ddH2O on the both plates and wipe off thoroughly with Kimwipes. Put the side spacers on the large plate. Wrap the bottom and sides with tape. 45. About 10 min from the end of the incubation, prepare the tubes for primer extension: thaw a 1 ml tube of extension mix in the 37oC water bath. Remove promptly after thawing and place on ice. Take the ice bucket to the enzyme

freezer and put the reverse transcriptase (Superscript II, BRL) and RNasin (Promega) into the ice bucket. 46. Invert the extension mix tube several times to mix. A small brown pellet stuck to the bottom is ok, but if there a significant amount of solution at the bottom of the tube that is darker than the rest of the solution, the extension mix has gone bad. 47. Pipet the following solutions into an empty screw-cap eppendorf tube:

# Rxns Extension Mix Rev Transcriptase RNasin 2-4 200 ul 3.3 ul 2.5 ul 5-8 400 ul 6.7 ul 5.0 ul 9-12 600 ul 10.0 ul 7.5 ul 13-16 800 ul 13.3 ul 10.0 ul

With the P200 set at "200", thoroughly mix the glycerol-laden proteins (Reverse Transcriptase; RNase inhibitor) into the extension mix by pipetting slowly at the bottom of the tube several times. Keep the prepared extension mix / RT / RNasin on ice. Put the RT and RNasin back into the enzyme freezer, and the rest of the extension mix back in your freezer. 48. After the 60 min annealing period is over, take the tubes out of the 45oC water bath, and spin 3s. Place the tubes in beta-blocks behind the hand-held shield. Unscrew the caps (leave them on the tubes). 49. With a different tip each time, pipet 41.5 ul of extension mix / RT / RNasin into each tube, and mix by pipetting slowly 3 times. Expel pipet tips into the waste container. Close the caps. 50. Put the tubes in the yellow holder, and incubate in the 45oC water bath for 60 min (put shield on bath again). Start the speed-vac. 51. During this time, take out a 60 cc syringe, spacer for sharktooth comb, 200 ul and 1000 ul pipet tips and their pipetters, and put them next to the two plates. Also obtain a 100 ml graduated cylinder and a 250 ml beaker and put them next to the plates. 52. Measure out 94 ml of 10% denaturing acrylamide (7M urea, 2X TEB) into a 100 ml graduated cylinder. Then pour into a 250 ml beaker. Return the acrylamide to the refrigerator. Take the TEMED and 10% APS (1g ammonium persulfate+ 10ml RNase-free water) solutions out of the refrigerator. Pipet 500 ul of the 10% APS solution into the beaker, then pipet 51 ul of the TEMED into the beaker. 53. Mix the above solution by swirling with the 60cc syringe, then draw up the solution into the syringe. Pick up one side of the gel at a 45o angle and squirt the acrylamide into one side. When the acrylamide is out of the syringe, put the plates face down on an empty tip box, and fill the plates to the top. Then put the square spacer for sharktooth comb in, first from one side, then the other. Do it carefully, or the acrylamide will splatter several inches. Then

immediately clamp the gel with 5 clamps on the top (all the way in), and 3 clamps on each side (halfway in). Continue to add drops of acrylamide to the gaps beside the spacer as needed to maintain a bead, until the acrylamide is completely polymerized (about 15 min.) Clean up everything carefully.

V. Termination of Primer Extension Reactions; Preparation of Samples for Gel Electrophoresis

54. About 10 minutes before the incubation time is over, prepare the tubes for phenol / chloroform extraction and ethanol precipitation: put a single line of screw-cap tubes in the rack(s), number them, and uncap them. Mix 63 ul of 0.3 M NaOAc with 7.5 ul of 3 M NaOAc / reaction tube (ex. 8 rxns. = 500 ul 0.3 M NaOAc and 60 ul 3 M NaOAc ex 10rxns= 630ul .3MNaOac, 75ul 3M NaOac). Mix by vortexing. 55. Put the rack(s) of tubes, 0.3 / 3 M NaOAc tube, phenol, chloroform, P200, and pipet tips into the hood. 56. When the 60 min incubation is over, take the tubes out of the 45oC water bath, spin down, and put them in the beta-blocks behind the small shield. Bring the tubes into the hood. Loosen the caps, and with one pipet tip add 200 ul of the phenol / chloroform mix to each tube. Then add 56 ul of the 0.3 / 3 M NaOAc mix into each tube. Tighten the caps. 57. Vortex up to eight tubes for 30s. Spin 3 min. If 9 or more tubes, vortex the remainder of the tubes not in the microfuge. 58. After the 1st group of tubes is finished spinning (if more than 8 tubes total), exchange tubes and spin 3 min. If not more than 8 tubes, take the tubes out of the microfuge and put them in the hood. 59. Put the tubes in the 3rd spaces in the racks and loosen the caps. Remove and save the aqueous phase of each tube by tilting the tube 45o and pipetting the top layer with a P200 Pipetman. Wait a few seconds after doing this (keep tube angle at 45o) to see the rest of the aqueous phase "comes around" to face you. Remove this also; being careful not to pick up any of the chloroform phase, and pipet into the new numbered but empty tubes. Remember to dispose of all caps, tubes, and pipet tips in the waste container. 60. Bring the tubes back to your bench, and add 300 ul cold 100% EtOH from your freezer. Close caps, vortex 30s, incubate in dry ice/EtOH bath for 15 min., then spin in cold room 15 min. Put the beta-blocks and shield just outside the cold room. 61. If running gel today, begin heating 400 ml of dH2O in a 2L beaker at full heat. Clean up everything, including in the hood. Run the Geiger counter over all objects before putting them back in your drawers. > 300 cpm = contaminated (put in beaker of dH2O with some Ivory soap and let sit for 1 day). 62. Carefully peel off the tape from the gel plates. Even more carefully take the square sharktooth spacer out: loosen the spacer at first by inserting a metal

spatula between the spacer and the plate at several places. Then, loosen the spacer away from the plate slowly by turning the spatula on each edge of the spacer so that it pushes out the spacer. Try to push each edge as evenly as possible. Wipe the liquid acrylamide off the plates and then remove the excess solidified acrylamide from the plates with a razor blade. Rinse the spacer with dH2O after removing it. 63. Obtain a large polyacrylamide gel apparatus, a plastic horizontal bar, and two blue rubbery spacers. Put the plates on the apparatus (large plate facing out). Make sure the plates are straight up and down (perpendicular to the bench) and then put the plastic bar over them. Put the blue spacers above the small plate (behind the long plastic spacers). Push them down tight with a metal spatula. Tighten the horizontal bar. 64. Mix 400 ml of 10X TEB with 1600 ml ddH2O in a 2 liter graduated cylinder (final = 2X TEB). Pour the TEB in the top and bottom reservoirs. Using a 5ml syringe with a curved needle, blow the excess acrylamide out of the spacer well and remove any bubbles from the bottom edge of the gel. 65. Take a curved large shield from a bench (the tall, kinked-at-the-top shield), and put it in front and under the gel apparatus by carefully tilting the apparatus and sliding the base of the shield under the apparatus. Put a temperature gauge in the middle of the front gel plate. Hook the electrodes to the power supply. Pre- warm the gel at 1800 volts (65 watts). Do not warm past 50oC or the plates may crack and ruin your gel. When the gel temp gets to 49oC, turn the watts down (it will take practice to know how long and how many watts to warm the gel). 66. Put a 50 ml conical tube in a 250ml beaker or styrofoam holder. 67. When the 15 min spin is complete, bring the tubes back to your bench and place behind shield. 68. Carefully pipet the ethanol out of the tube (twice) with the P200, the second time touching the bottom of the tube on the side opposite the pellet. Make sure you did not pick up the pellet on the pipet tip. Put the ethanol and pipet tips in the 50 ml conical tube. Put all waste in the radioactive trash. 69. Dry the samples by spinning in the Savant Speed-Vac for 2-3 min. [NOTE: The tubes should be placed in the speed vac without the caps on them] 70. With a P200, pipet out about 100 ul of 2x denaturing dye (loading buffer; denatures the DNA:RNA hybrid that is obtained after the Primer Extension Assay). With one pipet tip, pipet 4 ul of loading buffer onto the side of each tube. Close the tubes, and tap them to put the dye at the bottom. 71. Vortex the tubes normally 30s each, then individually vigorously vortex (on the side of the vortexer) for 5-30s, in order to remove the pellets from being stuck on the bottom of the tubes. Spin 5s. 72. If running gel today, skip to step 74. If not, put the tubes in the yellow holder, and put the holder and the small shield in your freezer overnight. Tomorrow go to step 74.

VI. Gel Electrophoresis and Autoradiography

73. If you did the experiment the night before, take out the small shield and tubes from the freezer. Thaw them briefly in the 37oC water bath, and wipe off the condensation from the shield. Vortex tubes 30s, then individually 5-30s each on the side of the vortex. Spin 5s. Prepare an ice-water bath in an insulated bucket. 74. When the H2O is boiling, put the reaction tubes in the yellow holder and put the holder in the boiling water for 2 min. 75. During this time, check the gel temperature and adjust accordingly (the temp. should be about 50oC when you load the samples). After 2 min. of boiling, take the yellow holder out of the beaker and immediately put it in the ice-water bucket for 90s. Take the tubes out of the ice and place in numerical order in beta-blocks behind the small shield. 76. Stop the gel, and blow out the urea from the well, insert the sharkstooth comb just into the gel, (hopefully the temp. is at 50oC; don't worry when it falls as you are loading your samples). 77. Place a large Kimwipe next to the gel apparatus, along with a P10 pipet and pipet tips. Load one lane with 4ul of loading dye only, and wait a few seconds to make sure the samples won't leak out of the lanes 78. Pick a good spot to load the first sample (middle-left). Load 4ul sample into the well without loading any air bubble if possible. 79. After all samples are loaded, wait a few seconds to let all the samples settle equally to the bottoms of the wells. Turn on gel again, this time to 65 watts. Clean up radioactive waste, and remember timer, ice bucket, yellow tube holder, and hot beaker. 80. Check the temperature of the gel every 10-20 min, maintaining it at 50oC. The gel should be run until the fast dye reaches 27 cm from the bottom of the wells. 81. About 5 min before the stopping the gel, cut out a 6" by 8" piece of filter paper. Also bring from your bench a pair of scissors, a spatula, squirt bottle with water and the radioactive waste container. Have Saran Wrap nearby. 82. Turn off the power to the gel, remove wires and put the power supply away. Take off the temperature gauge from the plate. Empty the top reservoir of most of its buffer with a 250 ml beaker. Unscrew the horizontal plastic bar, and lay the gel small plate up. 83. With a metal spatula, pry the smaller glass plate off of one corner. Lift carefully. If the gel sticks to the small plate, turn both plates over and then remove the large plate. 84. Put the filter paper on the gel over the reaction lanes. The bottom of the paper should be about half way between the fast and slow dyes. With a razor blade cut the gel around the edge of the paper. Squirt water around the paper where the edges of the gel are, and make a big X across the middle of the paper with the water. Press down only on the sides and bottom of the gel (1/2 inch perimeter). With the top (lanes) facing toward you, push down on the top

(inward 1 inch). Then slowly peel of the filter paper and gel, from the top to the bottom. 85. Cover the gel with a piece of Saran Wrap. Then trim the saran wrap, leaving 1/2-inch space on both sides up to the top of the gel. Very carefully fold the saran wrap under the edges of the gel/paper. 86. Take the Saran Wrap covered gel to the gel dryer and dry 20-40 min at 80oC. You can tell when the gel is dry when the filter paper is firm and doesn't sag when held up. Meanwhile, clean up everything else (put radioactive waste in lab's large waste container, and use Geiger counter on all objects, including bench). 87. When the gel is dry, put it into a labeled large metal cassette that has an intensifying screen. Put on film in the dark room, and place in the -80oC freezer for at least 12 hours.

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Appendix C. Detailed Protocol for Immuno-depletion Assay

I. Solutions and Materials 1. Pierce Immobilized Protein A/G Plus agarose resin (Capacity: > 50mg human IgG/mL of resin) 2. Anti-sera (Stored at -20°C): a. TBP antiserum (#75; 2/6/06) b. TRF1 antiserum (#78; 2/6/06) c. TBP antiserum + TRF1 antiserum d. Pre-Immune Serum 3. Binding buffer [(A/G) IgG Binding Buffer (Product No. 54200) from Pierce] 4. SNF wash buffer (Store at 4°C) Make 100ml of SNF pre-buffer with 135 mM KCl, 20mM HEPES, K+, 8.75 mM MgCl2, 0.1 mM EDTA, 10% Glycerol and distilled water using the table below (using highlighted cells of the table). Store at 4°C. TABLE 1 Reagents Stock Amount Final Concentration Solution Needed KCl 4M 3.375 ml 135 mM HEPES, K+ 1M, pH 7.6 2 ml 20 mM

MgCl2 1M 875 ul 8.75 mM EDTA 0.5M, pH 8.0 20 ul 0.1 mM Glycerol 100% 10 ml 10% DTT 1M 175 ul 1.75 mM PMSF 100mM 150 ul 0.15 mM Benzamidine 0.5M 200 ul 1 mM Na Metabisulfite 0.5M 200 ul 1 mM Water 83.205 ml Total 100ml NOTE: PMSF [PMSF is inactivated in aqueous solutions, so stock solution should be made in ethanol/isopropanol] and DTT stock solutions should be

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made ahead of time and stored in the freezer while Benzamidine and sodium metabisulfite should always be freshly prepared and added to the pre-buffer right before using the SNF wash buffer (in Step 6b).

Make 2 ml of SNF wash buffer by using the table below: TABLE 2 Reagents SNF pre- DTT PMSF Benzamidine Na buffer metabisulfite Amount 2 ml 3.5 3 ul 4 ul 4 ul ul

4M KCl Dissolve 149.1g of KCl in 300ml of distilled water and adjust the volume to 500ml. Sterilize by autoclaving and store at room temperature.

1 M HEPES, K+, pH 7.6 (100 ml) Add 23.83 g of HEPES to about 60 ml of dH2O in a 150 ml beaker. Add a stir bar, place on a stir plate, and stir. Slowly add solid KOH until the pH is 7.6. Pour the solution into a 100 ml graduated cylinder and add dH2O to the 100 ml mark. Store in freezer.

1M MgCl2 Dissolve 203.3 g (2.033 g) of MgCl2.6H2O in 800 ml (8 ml) of ddH2O. Adjust the volume to 1 L (10 ml) with ddH2O. Dispense into aliquots in a brown screw capped bottle and sterilize by autoclaving.

1M Dithiothreitol (200 ul) Dissolve 30.86 mg (0.03086 g) DTT (in freezer of the yellow fridge) to 200 ul of RNase-free water. Store at -20°C.

100 mM PMSF (Phenylmethylsulfonyl Fluoride) Also called α-toluenesulfonyl chloride, PMSF (m.w. 174.19) is used as an inhibitor of serine proteases. It is very toxic. Aqueous solutions of PMSF are hydrolyzed very rapidly. To make a 100mM stock solution, add 174mg (0.174g) of PMSF in 10ml absolute ethanol. Divide into aliquots and store in foil-wrapped tubes at -20°C. Sterilization is not required. Only add to aqueous solutions immediately before their use. CAUTION: PMSF is inactivated in aqueous solutions. The rate of inactivation increases with pH and is faster at 25°C than at 4°C. The half-life of a 20uM aqueous solution of PMSF is about 35 minutes at pH 8.0. This means that

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aqueous solutions of PMSF can be safely discarded after they have been rendered alkaline (pH.8.6) and stored for several hours at room temperature.

0.5 M Benzamidine Dissolve 0.157g of benzamidine in distilled water and adjust the volume to 2ml. Divide into aliquots and store at -20°C.

0.5 M Na metabisulfite Dissolve 0.19g of Na metabilsulfite in 2ml of distilled water. Store at room temperature.

II. Protocol This protocol involves 2 major steps: A. Binding of antibodies to immobilized Protein A/G plus agarose resin (This protocol does not involve cross-linking of the antibodies to the resins). This will be done for both TBP antiserum and TRF1 antiserum. 1. Equilibrate the Pierce Ultra-link Immobilized Protein A/G Plus resin (Capacity: > 50mg human IgG/mL of resin) and all buffers (binding and wash buffer) to room temperature. 2. Thaw the antibodies (TBP and TRF1 anti-sera), mix by gentle finger flicking and place them on ice. 3. Prepare an antiserum to be purified with the prepared Protein A/G Plus settled resin. a. Dilute the antiserum with the binding buffer in a 1:2 ratio (For Example, add 600 ul of the binding buffer to 300 ul of the antiserum to make a total of 900 ul of the diluted antiserum). [Note: 300 ul of antiserum should contain about 3-4.5 mg IgG]. b. Centrifuge this diluted antiserum at 10,000 g, 4°C for 20 minutes (EPPENDORF, Centrifuge 5415D in the cold room) to remove any particulates and cloudiness. [Note: Plasma may become hazy upon dilution with the binding buffer because of lipoprotein precipitation. Centrifugation needs to be done at 4°C because very high speed used for removing the cloudiness may raise the temperature of the antisera]. c. Pipet off the 900 ul of supernatant into a fresh tube at room temperature. 4. Prepare Immobilized Protein A/G Plus agarose settled resin. [INSTRUCTIONS: This can be done during the 20 min time duration while centrifuging the antisera (Step 3b)]

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a. Gently swirl the bottle/vial of Protein A/G Plus resin to obtain an even suspension. b. Using a P-1000 with ≈2mm cut-off end of the tip, immediately transfer 200 ul suspended resin (50 % slurry; 100 ul packed resin) from the vial to a chilled 1.5 ml screw-cap tube. [Note: Always use tips cut off at the end to handle the resin]. [Note: 100 ul packed resin is capable of binding about 5 mg IgG] c. Centrifuge at 2500 g for 3 minutes at room temperature. d. Pipet off the supernatant. e. Wash the resin three times with 400 ul of the binding buffer [(A/G) IgG Binding Buffer (Product No. 54200) from Pierce] to obtain a settled resin. i. Using a P-1000, add 400 ul of the binding buffer to the resin. ii. Re-suspend the beads by finger flicking or gentle vortexing. iii. Centrifuge at 2500 g for 3 minutes at room temperature. iv. Pipet off the supernatant. v. Repeat steps i-iv for two more times. 5. Incubate the antiserum with the resin to obtain a resin-linked IgG fraction (contains small fractions of TBP and TRF1) to be used for the immuno- depletion of the SNF. a. Using a P-1000, add the prepared 900 ul of the diluted antiserum (Step 3) to the 100 ul prepared settled resin (Step 4). b. End-over-end rotate at room temperature for 1 hr. [INSTRUCTIONS: About 10min before the incubation period is over, make 0.5 M Benzamidine (place on ice) and 0.5 M Na metabisulfite solutions]. c. Centrifuge at 2500 g for 3 minutes at room temperature. d. Pipet off and save the supernatant in another eppendorf tube. 6. After the 1 hr incubation, wash the resin thoroughly in order to remove any unbound serum proteins from the resin. a. Wash the resin 3 times with the binding buffer at room temperature. i. Using a P-1000, add 400 ul of the binding buffer and resuspend the beads by finger flicking or gentle vortexing. ii. End-over-end rotate for 5 minutes. iii. Centrifuge at 2500 g for 3 minutes. iv. Pipet off the supernatant (save the washes in other chilled screw-capped tubes at -20°C to run on SDS gels). v. Repeat steps i-iv for two more times. [NOTE: During the 5 min rotation period of the 3rd wash, take out DTT and PMSF from the -20°C freezer and prepare the SNF wash buffer (using table 2). Place it on ice].

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b. Wash the resin 2 times with the SNF wash buffer at 4°C. Work in the cold room with EPPENDORF Centrifuge 5415 D. [Note: When we wash resins, even if we remove the supernatant, there is going to be an aqueous solution in the resins because the beads are porous. Hence we need to wash the resins with SNF wash buffer (that contains all the chemicals used to make SNF) before incubating it with the SNF (Step 9) in order to keep the SNF in the buffer having similar chemical composition]. [Note: From this step, work in the cold room] i. Using a P-1000, add 400 ul of the SNF wash buffer and resuspend the beads by finger-flicking or gentle vortexing. ii. End-over-end rotate at 4°C for 5 minutes. iii. Centrifuge at 2500 g, 4°C for 3 minutes. iv. Pipet off the supernatant (save the washes in other chilled screw-capped tubes at -20°C to run on SDS gels). v. Repeat steps i-iv for 1 more time. [INSTRUCTIONS: During the 5 min rotation period of the last wash, thaw a tube of SNF and place it on ice].

B. Immuno-depletion of the SNF

7. Re-suspend the thawed tube of SNF by finger flicking. 8. To each 100 ul of settled antibody affinity resin (α-TBP and α-TRF1), equilibrated in the SNF buffer, add 400 ul of this thawed SNF and resuspend by finger flicking or gentle vortexing. [NOTE: The SNF gets more diluted with the increasing amount of the beads used for its immuno-depletion. 100ul of beads for immuno-depleting 400ul of SNF is a good ratio to use] 9. End-over-end rotate both of the samples (with α-TBP and α-TRF1) at 4°C for 4 hrs. 10. After the 4 hr incubation time is over, centrifuge the samples at 2500 g, 4°C for 3 minutes using EPPENDORF, Centrifuge 5415D in the cold room. 11. Save the supernatant containing the immuno-depleted SNF (about 400 ul) for each sample. Divide each SNF into 50 ul aliquots (enough for 3 reactions) distributing into 8 chilled screw-capped tubes. 12. Store the aliquoted immuno-depleted SNF in liquid nitrogen to be used in the in-vitro transcription assay. 13. Wash the beads twice with IgG binding buffer a. Using a P-1000, add 400 ul of the binding buffer and re-suspend the beads by finger flicking or gentle vortexing. b. Centrifuge at 2500 g for 3 minutes.

105 c. Pipet off the supernatant. d. Repeat steps a-c for one more time. e. Store the beads (with IgG bound) at 4°C.

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Appendix D. Detailed protocol for Nickel-chelate chromatography

I. Solutions and materials

O.1M CuSO4

Add 0.25 g CuSO4·5H2O into 9.91 ml sterile d.d. water in a 15 ml Falcon tube. Votex to dissolve. Working in the cell culture hood, use 0.22 µm pore-size syringe filter to filter/aliquate CuSO4 solution into 10 of 1.5ml conical screw-cap tubes (~1ml/tube). Store at 4°C.

Stock solution A (for making 5X Native purification buffer)

250 mM NaH2PO4 (monobasic sodium phosphate), 2.5 M NaCl

Dissolve 7.8 g NaH2PO4·2H2O (MW 155.99) and 29.2 g NaCl in 200 ml d.d. water. Filter to sterilize. Store at 4°C.

Stock solution B (for making 5X Native purification buffer)

250 mM Na2HPO4 (dibasic sodium phosphate), 2.5 M NaCl

Dissolve 7.1 g Na2HPO4 (MW 141.96) and 29.2 g NaCl in 200 ml d.d. water. Filter to sterilize. Store at 4°C.

3M Imidazole

Add 10.2 g imidazole into 30 ml sterile d.d. water in a 50 ml Falcon tube. Votex to dissolve. Fill with sterile d.d. water to 50 ml. Invert to mix. Store at 4°C.

5X Native purification buffer

Add 45 ml stock solution B in a 100 ml beaker with a stirring stir bar. Titrate with stock solution A (drop by drop, only very small amount is needed) until the pH reaches 8.0. Transfer the solution to a 50 ml Falcon tube and store at 4°C.

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1X Native purification buffer (for making Native binding buffer and Native wash buffer) (100 ml/purification)

Add 20 ml 5X Native purification buffer and 75 ml sterile d.d. water in a 150 ml beaker with a stirring stir bar. Titrate with HCl or NaOH until the pH reaches 8.0 (~11 µl of 12M HCl). Bring the volume to 100 ml with sterile d.d. water. Store at 4°C.

Native binding buffer w/ 10 mM Imidazole (12 ml/purification)

Add 30 ml 1X Native purification buffer and 100 µl 3M Imidazole in a 100 ml beaker with a stirring stir bar. Titrate with HCl or NaOH until the pH reaches 8.0 (~3 µl of 12M HCl). Transfer the solution to a 50 ml Falcon tube and store at 4°C.

Native wash buffer w/ 20 mM Imidazole (12 ml/purification)

Add 50 ml 1X Native purification buffer and 335 µl 3M Imidazole in a 100 ml beaker with a stirring stir bar. Titrate with HCl or NaOH until the pH reaches 8.0 (~15 µl of 12M HCl). Transfer the solution to a 50 ml Falcon tube and store at 4°C.

======Chemicals for preparation of HEMG-100 buffer:

1M HEPES K+ (pH 7.6)

Dissolve 119.15 g HEPES (MW 238.3) in 500 ml d.d. water to make 1M HEPES. Titrate with 10N KOH until the pH reaches 7.6. Store at 4°C.

1M MgCl2

Dissolve 101.65 g MgCl2·6H2O in 400 ml d.d. water. Adjust the volume to 500 ml with d.d. water. Filter or autoclave to sterilize. Store at 4°C.

Note: MgCl2 is extreamly hygroscopic. Buy small bottles and do not store opened bottles for long periods of time. Once the crystals become saturated with water, dispose of the chemical properly.

10mM ZnCl2

Dissolve 681.4 mg ZnCl2 in 500 ml sterile d.d. water. Filter to sterilize. Store at room temperature.

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0.5M EDTA (pH 8.0)

Add 90.8 g of Na2EDTA·2H2O to about 400 ml of d.d. water. Stir and adjust the pH to 8.0 with NaOH (~20g of NaOH pellet). Bring the volume to 500 ml with d.d. water if necessary. Strerilize by autoclaving. Store at room temperature.

Note: Na2EDTA·2H2O will not be dissolved until the pH of the solution is adjusted to approximately 8.0 by the addition of NaOH.

4M KCl

Dissolve 149.1 g KCl in 400 ml of d.d. water. Bring the volume to 500 ml with d.d. water. Strerilize by autoclaving. Store at room temperature.

1 M DTT (dithiothreitol)

Add 1.54 g DTT into 10 ml d.d water in a 15 ml Falcon tube.Vortex to dissolve. Wrap with aluminum foil and store at -20°C.

100 mM PMSF (phenylmethylsulfonyl fluoride)

Add 696 mg PMSF in a 50 ml Falcon tube. Add 30 ml 100% ethanol. Vortex to dissolve. Fill with 100% ethanol to 40ml. Invert to mix. Wrap the tube with aluminum foil and store at -20°C.

Note: PMSF is very toxic so handle with care. Aqueous solutions of PMSF are hydrolyzed very rapidly, so the stock solution needs to be made with absolute ethanol or 2-propanol and only add PMSF to aqueous solutions immediately before their use.

======

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HEMG-100 buffer (w/ 20mM Imidazole) (4 ml/purification)

original solution final solution Chemical conc. unit add unit conc. unit HEPES K+ (pH7.6) 1.0 M 500.0 µl 25.00 mM MgCl2 1.0 M 250.0 µl 12.50 mM ZnCl2 10.0 mM 20.0 µl 0.01 mM EDTA (pH8.0) 0.5 M 4.0 µl 0.10 mM KCl 4.0 M 500.0 µl 100.00 mM Glycerol 100.0 % 2.0 ml 10.00 % Imidazole 3.0 M 133.2 µl 20.00 mM PMSF 0.1 M 100.0 µl 0.50 mM Sterile d.d. water 16432.8 µl final volume 20.0 ml

Note: Add everything except DTT and PMSF into a 50 ml Falcon tube. Vortex to mix. Store at 4°C. Add DTT and PMSF right before use.

Elution buffer (HEMG-100 buffer w/ 750mM Imidazole) (3 ml/purification)

original solution final solution Chemical conc. unit add unit conc. unit HEPES K+ (pH7.6) 1.0 M 500.0 µl 25.00 mM MgCl2 1.0 M 250.0 µl 12.50 mM ZnCl2 10.0 mM 20.0 µl 0.01 mM EDTA (pH8.0) 0.5 M 4.0 µl 0.10 mM KCl 4.0 M 500.0 µl 100.00 mM Glycerol 100.0 % 2.0 ml 10.00 % Imidazole 3.0 M 5.0 ml 20.00 mM PMSF 0.1 M 100.0 µl 0.50 mM Sterile d.d. water 11566.0 µl final volume 20.0 ml

Note: Add everything except DTT and PMSF into a 50 ml Falcon tube. Vortex to mix. Store at 4°C. Add DTT and PMSF right before use.

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Dialysis buffer (HEMG-100 buffer W/O imidazole)

original solution final solution Chemical conc. unit add unit conc. unit HEPES K+ (pH7.6) 1.0 M 50.0 ml 25.00 mM MgCl2 1.0 M 25.0 ml 12.50 mM ZnCl2 10.0 mM 2.0 ml 0.01 mM EDTA (pH8.0) 0.5 M 400.0 µl 0.10 mM KCl 4.0 M 50.0 ml 100.00 mM Glycerol 100.0 % 200.0 ml 10.00 % DTT 1.0 M 6.0 ml 3.00 mM PMSF 0.1 M 10.0 ml 0.50 mM Sterile d.d. water to 2L final volume 2.0 L

Note: Add everything except DTT and PMSF into a 4L plastic beaker with 1.5 L d.d. water. Stir to mix. Bring the volume to 2L with d.d. water. Stir to mix. Store at 4°C. Add DTT and PMSF right before use.

DPBS (Invitrogen, product code 14190) (Dulbecco’s PBS)

Note: You can use any other 1X PBS from other vendors.

Protease Inhibitor Cocktail (SIGMA, product code P8340)

CelLytic M Lysis buffer (SIGMA, product code C2978)

ProBond Nickel-Chelating Resin (Invitrogen, product code 46-0019)

Poly-Prep Chromatography columns (BioRad, product code 731-1550)

Spectra/Por 2 dialysis tubing (MWCO 12-14000 Da, nominal flat width 10 mm, 0.32 ml/cm) (SpectrumLab, product code 132676)

Dialysis tubing clamps

II. Expression of DmSNAPs in S2 cells

1. Grow 8 big plates (Corning 100 x 20 mm tissue culture plates) of cells in 20 ml selective medium to 70-80% confluency.

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2. Induce cells with copper sulfate. Add 100 µl 0.1M CuSO4 into each plate (to final 0.5 mM). 3. Incubate cells for ~24 hours at 22-25°C.

III. Preparation of ProBond column

1. Put a poly-prep chromatography column in a 50ml Falcon tube. 2. Thoroughly suspend the ProBond resin in the vial in order to make a uniform suspension of the resin. Ratio of volume of suspension to packed gel is 2 to 1. 3. Using a 10 ml serological pipet. Immediately transfer 2 ml of suspended resin (containing 1ml packed resin) into the chromatography column. 4. Cap the column. Centrifuge the resin for 1 min at 420 g. 5. Remove the supernatant. Be careful not to remove any resin. 6. Wash the resin once in sterile d.d. water. a. Add 6 ml sterile d.d. water to the column. Resuspend the resin thoroughly by inverting/tapping the column. b. Cap the column. Centrifuge at 420 g for 1min. c. Remove the supernatant. 7. Wash the resin twice with Native binding buffer. a. Add 6 ml Native binding buffer to the column. Resuspend the resin thoroughly by inverting/tapping the column. b. Cap the column. Centrifuge at 420 g for 1min. c. Remove the supernatant. d. Repeat steps a-c.

Note: For the last wash with Native binding buffer, if the resin will not incubate with cell lysates immediately, stop at Step a, leave the resin suspended in Native binding buffer and keep the column in the cold room. Centrifuge to remove the supernatant right before use.

IV. Preparation of dialysis tubing

1. Cut the dialysis tubing into 8cm pieces (the capacity is ~ 1ml/piece). 2. Put the tubing pieces one-by-one into a beaker containing d.d. water with magnetic stir bar stirring. Allow it to stir for 30 mins. Make sure all pieces completely immersed in the water during stirring. 3. Transfer the tubing pieces into another beaker containing d.d. water. Make sure all pieces completely immersed in the water. Cover the beaker with aluminum foil and keep it in the cold room until used.

V. Procedures of His-tagged protein purification

A. Cell lysis

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1. Add and mix 70 µl protease inhibitor cocktail (to final 1%) and 23.3 µl 3M imidazole (to final 10 mM) to 7 ml of chilled CelLytic M lysis buffer in a 15 ml Falcon tube. Keep the tube on ice. 2. Harvest cells: (from 8 big plates) a. To remove cells adhering to the dish, pipet the medium over the cells to wash cells off the plate for several times. b. Collect the cells and medium from every 2 plates to one 50 ml centrifuge tube. (So for each cell line you will need 4 tubes for the 8 big plates) c. Centrifuge in the RT-Legend at 420 g for 5 min. d. Suck out the supernatant and discard. e. Wash the cells by resuspending the pellet in one 50 ml tube with 10 ml of DPBS. Transfer the suspended cells to another pellet-containing 50 ml tube. Pipet up and down to resuspend the pellet thoroughly. Now all our cells are in a single 50 ml tube. f. Centrifuge in the RT-Legend at 420 g for 5 min (use balance tube if necessary). g. Suck out the supernatant and discard. 3. Lyse cells: a. Transfer all 7ml of CelLytic M lysis buffer supplemented with 1% protease inhibitor and 10mM imidazole (Step IV-1) to the 50 ml tube containing washed cell pellet (Step V-2-g). Pipet up and down until all cells are lysed. b. Transfer the lysed cells back to the 15 ml tube used for CelLytic M lysis buffer storage. Keep the 15 ml tube on ice and move to the cold room. 4. In the cold room, rotate the 15 ml tube containing lysed cells end-over-end for 15 mins to ensure complete lysis. Note: From now on, you need to handle the lysed cells in the cold room. 5. After the 15 min incubation, aliquot the lysed cells into 7 of chilled 1.5 ml conical screw-cap tube (~1ml/tube). Centrifuge the cell lysate 12,000g for 10 mins in the microfuge in the cold room (EPPENDORF, Centrifuge 5415D). 6. Pool the supernatant from the 7 tubes (~7 ml total) into a new chilled 15 ml Falcon tube. Place the tube on ice. 7. If the lysate is viscous, shear the DNA by passing it through an 18-gauge needle four times. a. Put an 18-gauge needle on a 10 ml syringe. b. Suck the lysate into the syringe and expel it back into the tube slowly four times. 8. Remove 100 µl of the lysate into a chilled 1.5 ml conical screw-cap tube labeled as “lysates” and freeze in liquid N2. Keep the remainder of lysates on ice.

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9. Measure the total amount of the remaining lysate. Add calculated amount of 5M NaCl into lysate to give a final concentration of 500 mM NaCl. Note: Add 105.1 µl of 5M NaCl per ml of lysate to get final concentration of 500mM NaCl. For example, if the volume of lysate is 7 ml, add 735.7 µl of 5M NaCl.

B. Resin binding

1. In the cold room, add the cell lysates (~7 ml) to the prepared column containing packed ProBond resin (Step III-7). Resuspend the resin thoroughly by inverting/tapping the column. 2. Incubate the lysates and resin for 2 hrs in the cold room on the rocker Note: The incubation time may need to be optimized if proteins other than DmSNAPs are purified. For Bdp1 (or any other protein containing Bdp1) purification, incubate only for 30 min 3. Centrifuge the resin for 1min at 420 g in the RT-Legend. Transfer the supernatant into a chilled 15 ml Falcon tube. Remove 100 µl of the supernatant from the tube into a chilled 1.5 ml conical screw-cap tube and store frozen in the liquid nitrogen as “Flowthrough”. Store the remainder in -80°C. 4. Wash the resin three times with Native wash buffer. a. Add 4 ml Native wash buffer to the column. Resuspend the resin thoroughly by inverting/tapping the column. Note: Add 40 ul protease inhibitor cocktail for Bdp1 (or any other protein containing Bdp1) purification b. Cap the column. Centrifuge at 420 g for 1min. c. Remove the supernatant. a. Repeat steps a-c for two more times.

5. Wash the resin once with HEMG-100 buffer (w/ 20 mM Imidazole). a. Add 4 ml HEMG-100 buffer (w/ 20mM Imidazole) to the column. Resuspend the resin thoroughly by inverting/tapping the column. Note: Add 40 ul protease inhibitor cocktail for Bdp1 (or any other protein containing Bdp1) purification b. Cap the column. Centrifuge at 420 g for 1min. c. Remove the supernatant.

C. Elution of the His-tagged protein

1. Elute the bound His-tagged protein with three 1ml volumes of Elution buffer (HEMG-100 buffer w/ 750 mM Imidazole). a. Clamp the column in a vertical position and snap off the cap on the lower end. Allow the remainder of the buffer in the column flow out and discard (will be just few drops).

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b. Place a chilled 15 ml Falcon tube under the column. Position the tube to ensure that in next step, every drop of the elution from the column will be collected by the 15 ml tube. c. Add 1ml of the elution buffer to the column drop by drop. Allow the eluted proteins to come out into the 15 ml tube underneath the column until completely drained (about 5mins). d. Add 3 ul of 1M DTT to each 1 ml elution fraction. e. Remove 50 µl of the elution fraction into a chilled 1.5 ml conical screw-cap tube and store frozen in the liquid nitrogen as “Elution 1”. Save the remainder in the tube (~950 µl) on ice. f. Repeat steps b-d twice to collect fractions 2 and 3.

2. Now you should have three 15 ml Falcon tubes sitting on ice as Elution 1, 2, and 3.

VI. Dialysis to remove imidazole in elution fractions

1. Prepare a 2L beaker containing 1L of ice-cold dialysis buffer (HEMG-100 buffer W/O imidazole) with stirring. Add 3ml of 1M DTT and 5ml of 100mM PMSF. Stirring. 2. Take out a prepared dialysis tubing (Step IV-3). Remove all the water remained inside/outside of the tubing. Use a dialysis clamp to close one end of the tubing. Pipet the Elution 1 from Step V-C-2 into the tubing. Close the other end with another clamp. Put the clamped tubing into the beaker containing dialysis buffer with stirring. Make sure the tubing is completely submerged. 3. Repeat Step 1 and 2 twice to transfer Elution 2 and 3 into individual dialysis tubing. Allow samples to dialyze for 2hr. 4. Exchange the dialysis buffer in the beaker with another 1L of ice-cold dialysis buffer supplemented with DTT and PMSF. Dialyze for another 2 hr. 5. Take out the tubing containing Elution 1. Unclamp one end of the tubing. Transfer the solution into a chilled 1.5 ml conical screw-cap tube labeled with “Elution 1 dialyzed”. Store frozen in the liquid nitrogen. 6. Repeat Step 5 twice to transfer Elution 2 and 3 into individual 1.5 ml conical screw-cap tubes as “Elution 2 dialyzed” and “Elution 3 dialyzed”. Store frozen in the liquid nitrogen.

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Appendix E. Detailed Protocol for electrophoretic mobility shift assay (EMSA)

I. Solutions and materials

Materials for preparation of radioactive DNA oligo probes:

Annealed double stranded DNA oligos (PSEAs) (1 µg/5 µl)

T4 polynucleotide kinase (T4 PNK) (NEB, product code: M0201L)

10x PNK reaction buffer (reagent supplied with T4 PNK)

32P gamma-ATP (3000 Ci/mmol, 10m Ci/µl) (PerkinElmer)

Chloroform/isoamyl alcohol (24:1)

Saturated phenol

Quick Spin Columns for radiolabeled DNA purification Sephadex G- 25, fine (ROCHE, product code: 11273949001)

Materials for bandshift/supershift reactions:

Radioactive oligo probes

0.1M DTT (dithiothreitol)

Diluted from 1M DTT. Preparation of 1M DTT is described two pages ahead.

Poly(deoxyguanylic-deoxycytidylic) acid sodium salt [Poly (dG-dC)] (SIGMA, product code: P9389)

Dissolved in HEMG-100 buffer to obtain final concentration of 1µg/µl.

Poly(deoxyinosinic-deoxycytidylic) acid sodium salt [Poly (dI-dC)] (SIGMA, product code: P4929)

Dissolved in HEMG-100 biffer to obtain final concentration of 1µg/µl.

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Glycerol

Purified proteins (DmSNAPs)

Appropriate antibodies (for supershift reactions)

HEMG-100 buffer

original solution final solution Chemical conc. unit add unit conc. Unit HEPES K+ (pH7.6) 1.0 M 500.0 µl 25.00 mM MgCl2 1.0 M 250.0 µl 12.50 mM ZnCl2 10.0 mM 20.0 µl 0.01 mM EDTA (pH8.0) 0.5 M 4.0 µl 0.10 mM KCl 4.0 M 500.0 µl 100.00 mM Glycerol 100.0 % 2.0 ml 10.00 % Sterile d.d. water 16726.0 µl final volume 20.0 ml

Note: Add everything into a 50 ml Falcon tube. Vortex to mix. Store at 4°C ======Chemicals for preparation of HEMG-100 buffer:

1M HEPES K+ (pH 7.6)

Dissolve 119.15 g HEPES (MW 238.3) in 500 ml d.d. water to make 1M HEPES. Titrate with 10N KOH until the pH reaches 7.6. Store at 4°C.

1M MgCl2

Dissolve 101.65 g MgCl2·6H2O in 400 ml d.d. water. Adjust the volume to 500 ml with d.d. water. Filter or autoclave to sterilize. Store at 4°C.

Note: MgCl2 is extreamly hygroscopic. Buy small bottles and do not store opened bottles for long periods of time. Once the crystals become saturated with water, dispose of the chemical properly.

10mM ZnCl2

Dissolve 681.4 mg ZnCl2 in 500 ml sterile d.d. water. Filter to sterilize. Store at room temperature.

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0.5M EDTA (pH 8.0)

Add 90.8 g of Na2EDTA·2H2O to about 400 ml of d.d. water. Stir and adjusted the pH to 8.0 with NaOH (~20g of NaOH pellet). Bring the volume to 500 ml with d.d. water if necessary. Strerilize by autoclaving. Store at room temperature.

Note: Na2EDTA·2H2O will not be dissolved until the pH of the solution is adjusted to approximately 8.0 by the addition of NaOH.

4M KCl

Dissolve 149.1 g KCl in 400 ml of d.d. water. Bring the volume to 500 ml with d.d. water. Strerilize by autoclaving. Store at room temperature.

1 M DTT (dithiothreitol)

Add 1.54 g DTT into 10 ml d.d water in a 15 ml Falcon tube.Vortex to dissolve. Wrap with aluminum foil and store at -20°C.

100 mM PMSF (phenylmethylsulfonyl fluoride)

Add 696 mg PMSF in a 50 ml Falcon tube. Add 30 ml 100% ethanol. Vortex to dissolve. Fill with 100% ethanol to 40ml. Invert to mix. Wrap the tube with aluminum foil and store at -20°C.

======Materials for preparation of non-denaturing polyacrylamide gel:

40% non-denaturing acrylamide stock solution (30:1)

Dissolve 38.71 g of electrophoresis-grade acrylamide, 1.29 g electrophoresis-grade bis-acrylamide in 100 ml d.d. water with stirring. Sterilize by passage through a 0.22-µm filter. Wrap the bottle with foil and store at 4°C. Discard the solution if the color turns yellow during storage.

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10x non-circulation buffer

Dissolve 60.58 g of Tris, 285.28 g of glycine, and 7.44 g of EDTA in 1.6 L d.d. water with stirring. Stir and adjust the pH to 8.3 with HCl. Bring the volume to 2 L with d.d. water. Store at 4°C (indefinitely). Discard the solution if the color turns yellow during storage.

1x non-circulation buffer (gel-running buffer)

Dilute from 10x non-crculation buffer with d.d. water. Around 800 ml is required for each gel-running apparatus.

10% Ammonia Persulfate (APS)

Add 1 g APS in a 15 ml Falcon tube. Add 10 ml sterile d.d. water. Vortex to dissolve. Aliquot the solution into microfuge tubes (1ml/tube). Store at - 20°C (indefinitely).

Note: APS provides the free radicals that drive polymerization of acrylamide and bis-acrylamide. APS decomposes gradually (it will last only a week at 4°C). Thus, once leave the freezer and get thawed, the 10% APS must stay on ice all the time and put back to the freezer right after use.

TEMED

Store at 4°C. Keep on ice when in use.

Note: TEMED serves as the catalyst for the polymerization of acrylamide and bis-acrylamide.

Large and small glass plates, spacers, vacuum glue, metal clamps, 20- well combs, non-stick reagent, pieces of sponge, gel-running apparatus, power supply and wires, food wrap, intensifier screen, film cassette

II. Preparation of Quick Spin columns

You will need to prepare two columns at the same time following the instruction below. 7. Place a collection tube with the end cut-off in a 15 ml Falcon tube. Do another set for the 2nd column. 8. Thoroughly suspend the G-25 resin in columns to make a uniform suspension of the resin by ticking/inverting the column.

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9. Remove the cap and tip from columns. Place the two columns into each 15 ml Falcon tubes prepared in step III-1. 10. Centrifuge in the RT-Legend at 1000 g for 3 min, 4°C. 11. Use forceps to take out the column for a while. Remove the flowthrough and the end-cut-off collection tube from the 15 ml Falcon tube. Replace the column and the end-cut-off collection tube back to the 15 ml Falcon tube. 12. Centrifuge in the RT-Legend at 1000 g for 3 min, 4°C. 13. Use forceps to take out the column for a while. Remove the flowthrough and the end-cut-off collection tube from the 15 ml Falcon tube. Replace the column and an intact collection tube back to the 15 ml Falcon tube.

Note: label one intact collection tube with “1”, and label the other with the name of your oligos and the date of radiolabeling. Put the column label with “1” into the 15 ml Falcon tube containing “1” collection tube, put the “2” column into the 15 ml Falcon tube containing the collection tube labeled with the detailed information of your oligos.

III. Radiolabeling of annealed DNA oligos

13. Prepare the following reaction in a 1.5 ml conical screw-cap tube:

1 µg annealed oligos (PSEAs) 5 µl 10x T4 PNK reaction buffer 5 µl T4 PNK (10 U/µl) 1 µl 32P gamma-ATP 8 µl Sterile d.d. water 31 µl final volume 50 µl

Note: use long P10 tips for transferring the gamma-ATP to avoid potential contamination.

14. Incubate the tube in a 37°C water bath for 30 mins. 15. While waiting, prepare the Quick Spin column as described in section II. 16. Take out the oligo tube from the water bath. Add 25 µl of chloroform/isoamyl alcohol (24:1) into the tube. 17. Add 25 µl of saturated phenol (get the lower layer) into the tube. 18. Vigorously vortex for 30 sec. 19. Centrifuge at 12000 rpm or maximum speed for 3 min at room temperature. 20. Transfer the aqueous top layer (about 50 µl) from the tube into the center of the prepared column labeled with “1” atop the “1” collection tube inside the 15 ml Falcon tube. 21. Centrifuge in the RT-Legend at 1000 g for 3 min, 4°C.

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22. Use forceps to remove the column and transfer the collection tube containing radiolabeled oligos to a rack. 23. Transfer the radiolabeled oligos (about 50 µl) into the center of the prepared column labeled with “2” atop the collection tube labeled with the detailed oligo information inside the 15 ml Falcon tube. 24. Centrifuge in the RT-Legend at 1000 g for 3 min, 4°C. 25. Use forceps to remove the column and transfer the collection tube containing radiolabeled oligos to a rack. Insert the removable cap to the tube. Now this is your radiolabeled and purified DNA oligos. Store your purified oligos on ice if using immediately, otherwise store at -20°C. 26. Prepare two 1.5 ml conical screw-cap tubes each containing 2 µl of purified radioactive oligos. Examine the radioactivity of oligos in these two tubes in the scintillation counter (use #6 for 32P). Store these two tubes in the -20°C freezer for future use. Note: to calculate the radioactivity (cpm) of your probes, if the result from the counting is “A” cpm from tube 1 and “B” cpm from tube 2, then the cpm/µl of your probe is (A cpm + B cpm)/(2 µl x 2) = (A+B)/4 cpm/µl

IV. Preparation of non-denaturing polyacrylamide gel

1. Apply non-stick reagents on a small glass plate. Use kimwipes to spread the reagent evenly. Assemble the treated small glass plate with a large glass plate and 3 spacers into a “sandwich” with vacuum glue applied at the joint of the spacers. Clamp the edge of the sandwich with metal clamps (2 on lower part of each right and left side, 3 on the bottom side. Total 7 clamps are used in this step). Lay the sandwich on a tip box on the bench so the sandwich is tilted with the bottom side touching the benchtop. 2. Prepare the 5% non-denaturing acrylamide solution as follows: a. Add 7.5 ml 40% non-denaturing acrylamide, 6 ml 10x non-circulation buffer, and 46.5 ml d.d. water into a 250 ml flask. Swirl to mix. Remove 5 ml from the gel solution and discard. b. Add 400 µl of 10% APS and 40 µl of TEMED into the flask. Swirl to mix (avoid bubbles). Use a transfer pipet to remove bubbles if necessary. Note: if you need to run supershift reactions on the gel, then you might need to prepare a 4% gel instead: mix together 5 ml of 40% non-denaturing acrylamide, 5ml of 10x non-circulation buffer, 39.56 ml of d.d. water, 400 µl of 10% APS and 40 µl TEMED in the flask. You don’t need to discard any gel solution in this case. 3. Immediately (but slowly and steady) pour the gel solution into the middle space of the assembled sandwich. Avoid any bubbles that may occur. Insert a 20-well comb and immediately clamp two extra metal clamps (one for each upper part of right and left side) to fix the comb. 4. Wait for 30 min allowing the gel to completely polymerize.

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5. Once the gel is solidified, hook up the gel sandwich onto a gel-running apparatus connected to a power supply with wires. 6. Remove the comb. Pour 1x non-circulation buffer into the upper tank and the lower tank of the gel-running apparatus so the wells of the gel are completely immersed in the buffer. Use a syringe with needle to remove unpolymerized acrylamide and bubbles inside wells, and to remove bubbles from the space in the bottom of the gel. 7. Run to warm up the gel at 100 V for 30 min. Note: Do not start this warm-up step until your bandshift/supershift reactions are ready for the 30 min incubation.

V. Preparation of bandshift/supershift reaction

1. Calculate how much radioactive probe you need according to the number of your reactions and the radioactive strength of the probe. Each reaction requires 1 µl of 20000 cpm/µl probe. For example, if you need 20 reactions and the radioacitivity of your probes measured from step III-14 is 400000 cpm/µl, then:

(20000 cpm/µl x 20 µl)/ (400000 cpm/µl) = 1 µl

Thus, you need 2.5 µl of the 400000 cpm/µl probe to dilute with (20-1=19) µl of d.d. water to make 20 µl of 20000 cpm/µl probe for your bandshift reaction. You can also use 22 µl instead of 20 µl in the equation to make sure you have enough probe to use. 2. Prepare the probe-mix by mixing 1 µl of the 20000 cpm/µl probe, 2 µl of 1 µg/µl poly (dI-dC) or poly (dG-dC), and 1 µl of 0.1 M DTT in a 1.5 ml conical screw-cap tube for each reaction (so 4 µl of probe-mix per reaction). Multiple by the number of your total reactions to see how much of each reagent you really need. 3. Prepare each bandshift reaction as follow:

HEMG-100 (15-X) µl Sterile d.d. water 2 µl Probe-mix 4 µl Proteins (DmSNAPs) X µl Final volume 21 µl

Note: the final salt concentration should be around 80 mM, and the final glycerol concentration should be around 8%.

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4. Incubate the reactions in a 20°C water bath for 30 mins. If a supershift reaction is included, add antibodies in the middle of the incubation (15 min after incubation). Start this step with step IV-7 (gel warm-up) at the same time.

VI. Gel running and autoradiography

1. Load each well of the gel with each of your bandshift/supershift reactions. Load a empty well on the side with the non-denaturing dye. Run the gel at 120 V until the fast dye is approximately 3/4 through the gel (it will take around 3 hrs). Note: you might need to run the gel longer to allow the dye close the bottom of the gel if you have supershift reactions. This will allow the protein-DNA bands to separate further then it will be much easier to observe supershift bands. 2. Detached the gel sandwich from the gel-running apparatus. Dissemble the sandwich to allow the gel to separate from the small glass plate but stay on the large plate. 3. Tilt the plate with the gel on it to allow the buffer remained on the gel to run away from the gel. Use kimwipes to absorb the buffer. 4. Immediately lay a piece of food wrap on the surface of the gel. That piece of food wrap needs to be large enough to cover the whole gel and the large glass plate to allow full wraping of the gel. 5. In a dark room, place your wrapped gel/glass plate in a film cassette. Place a film on top of the gel. Place an intensifier screen on top of the film. Close and tightly fasten the cassette. Put the cassette into a -80°C freezer to allow the exposure of the film up to 18 hrs. 6. Develop the film in the darkroom to see the result.