University of Iowa Iowa Research Online eses and Dissertations 2009 Regulated release of P-Te from the 7sk Snrnp Brian Krueger University of Iowa Copyright 2009 Brian J. Krueger is dissertation is available at Iowa Research Online: hps://ir.uiowa.edu/etd/839 Follow this and additional works at: hps://ir.uiowa.edu/etd Part of the Cell Biology Commons Recommended Citation Krueger, Brian. "Regulated release of P-Te from the 7sk Snrnp." PhD (Doctor of Philosophy) thesis, University of Iowa, 2009. hps://doi.org/10.17077/etd.n88s3gow.
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University of IowaIowa Research Online
Theses and Dissertations
2009
Regulated release of P-Tefb from the 7sk SnrnpBrian KruegerUniversity of Iowa
Copyright 2009 Brian J. Krueger
This dissertation is available at Iowa Research Online: https://ir.uiowa.edu/etd/839
Follow this and additional works at: https://ir.uiowa.edu/etd
Part of the Cell Biology Commons
Recommended CitationKrueger, Brian. "Regulated release of P-Tefb from the 7sk Snrnp." PhD (Doctor of Philosophy) thesis, University of Iowa, 2009.https://doi.org/10.17077/etd.n88s3gow.
__________________________________ Title and Department
__________________________________ Date
REGULATED RELEASE OF P-TEFB FROM THE 7SK SNRNP
by
Brian Krueger
A thesis submitted in partial fulfillment of the requirements for the Doctor of
Philosophy degree in Molecular and Cellular Biology in the Graduate College of
The University of Iowa
December 2009
Thesis Supervisor: Professor David H. Price
Graduate College The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
PH.D. THESIS
_______________
This is to certify that the Ph.D. thesis of
Brian Krueger
has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Molecular and Cellular Biology at the December 2009 graduation.
Thesis Committee: ___________________________________ David H. Price, Thesis Supervisor
___________________________________ John Colgan
___________________________________ Anton McCaffrey
___________________________________ Madeline Shea
___________________________________ Lori Wallrath
ii
ACKNOWLEDGMENTS
I would like to thank my thesis advisor Dr. David Price for his support and
guidance throughout my graduate study. Working with him has resulted tremendous
growth in me as a scientist and I appreciate all of the discussions we have had during my
graduate career.
I would also like to thank the current and past members of the Price Lab. My
original project came as a result of Sarah Byers and Qintong Li’s thesis work. I thank
them for their patience and support while teaching me all of the techniques of the Price
Lab. Stanley Sedore, Bo Cheng, and Yan Jiang were always nearby for entertainment. I
thank them for the many good laughs and stories we shared throughout the years. I
would also like to thank Courtney Searcey for her LARP7 EMSA work.
Finally, I’d especially like to thank Jeff Cooper and Kathy Varzavand. Jeff was
instrumental in helping me to express and purify the proteins and antibodies I used
throughout my thesis work. I thank Kathy for taking care of much of my cell culture
work and always having cells ready for me when I needed them.
iii
ABSTRACT
Regulation of transcription elongation by P-TEFb is critical for proper gene
expression and cell survival. The cell possesses large quantities of P-TEFb, but the vast
majority of it is inactive in the 7SK snRNP. The 7SK snRNP is composed of the small
nuclear RNA 7SK, an inhibitory protein HEXIM which mediates the interaction between
P-TEFb and 7SK, the Methyl Phosphate Capping Enzyme, and the 7SK stability protein
LARP7. Since the discovery of the 7SK snRNP, research has been conducted to
determine how P-TEFb is released from this complex. The goal of the research presented
in this thesis is to better understand how the 7SK snRNP regulates P-TEFb and
ultimately, gene expression.
This work documents the discovery and characterization of the 7SK stability
protein LARP7. LARP7 is is associated with 7SK regardless of the presence of P-TEFb
and HEXIM1. Stabilization of 7SK is essential for maintenance of the RNP because loss
of LARP7 results in an increase in free P-TEFb and a significant reduction in the amount
of 7SK. These results indicate that stabilization of the 7SK snRNP by LARP7 is
important for regulating P-TEFb homeostasis.
Although P-TEFb was first characterized from Drosophila lysates, the
conservation of the 7SK snRNP and the mechanisms regulating P-TEFb inhibition have
not been described. Here, the Drosophila melanogaster homologues of LARP7 and 7SK
are characterized. These studies show that the system of P-TEFb regulation is similar in
flies and this makes Drosophila an attractive model system for studying P-TEFb
regulation through embryonic and larval development.
Finally, factors and modifications involved in releasing P-TEFb directly are
explored. An assay was developed for discovering proteins that can bind to and release
P-TEFb from the 7SK snRNP. Use of this assay showed that phosphorylation,
dephosphorylation, and acetylation of the components of the 7SK snRNP do not cause P-
iv
TEFb release directly. However, HIV Tat and the C-terminal P-TEFb binding region of
the bromodomain containing protein, Brd4, are capable of extracting P-TEFb directly.
Most importantly, the release of P-TEFb is followed by a conformational change in 7SK
RNA that causes HEXIM1 to dissociate from the complex. P-TEFb release from the 7SK
snRNP is the result of direct extraction of P-TEFb by viral or cellular proteins, and not
post-translational modifications or a competition between HEXIM1 and hnRNP proteins
for 7SK binding.
v
TABLE OF CONTENTS
LIST OF FIGURES .......................................................................................................... vii LIST OF ABBREVIATIONS ............................................................................................ ix
CHAPTER 1 INTRODUCTION ........................................................................................1 The RNA Polymerases .....................................................................................2 Transcription Initiation .....................................................................................3 RNAPII Transcription Elongation Control .......................................................4 Transcription Termination and 3′ End Formation ............................................5 Regulation of P-TEFb by the 7SK snRNP .......................................................6 Gene Specific Regulation of Transcription by P-TEFb ....................................7 P-TEFb Regulation and Associated Diseases ...................................................9 Focus of the Thesis .........................................................................................10
CHAPTER 2 LARP7, A NOVEL COMPONENT OF THE 7SK SNRNP STABILIZES 7SK SNRNA IN HUMAN CELLS .........................................14 Introduction .....................................................................................................14 Materials and Methods ...................................................................................16
Generation and Affinity Purification of LARP7 Antibodies ...................16 Glycerol Gradient Sedimentation Analysis .............................................17 Western Blotting ......................................................................................17 Immunoprecipitation ...............................................................................18 Small Interfering RNA Knockdown of LARP7 and MePCE ..................19 Electrophoretic Mobility Shift Assay ......................................................19
Results .............................................................................................................19 LARP7 Co-sediments with the 7SK snRNP ...........................................19 LARP7 is a Stable Component of the 7SK snRNP .................................21 Knockdown of LARP7 Disrupts the 7SK snRNP ...................................22
CHAPTER 3 DISCOVERY AND CHARACTERIZATION OF THE DROSOPHILA MELANOGASTER 7SK SNRNP .......................................38 Introduction .....................................................................................................38 Materials and Methods ...................................................................................40
Generation of Affinity Purified Drosophila LARP7 Antibodies .............40 Glycerol Gradient Sedimentation Analysis .............................................41 Immunoprecipitation ...............................................................................41 Western Blotting ......................................................................................42 RNA Isolation and Northern Blotting .....................................................42 Conservation Analysis .............................................................................43
Results .............................................................................................................43 Identification of dLARP7 ........................................................................43 dLARP7 Co-sediments and Co-immunoprecipitates with CyclinT and dHEXIM in an RNase Sensitive Complex .......................................44 Characterization of d7SK and the Drosophila 7SK snRNP ....................46
CHAPTER 4 MECHANISM OF RELEASE OF P-TEFB FROM THE 7SK SNRNP ...........................................................................................................64 Introduction .....................................................................................................65 Materials and Methods ...................................................................................69
Expression and Purification of Recombinant Proteins ............................69 Release Assay ..........................................................................................70 Western Blotting ......................................................................................71 Chemical Modification of 7SK RNA ......................................................71 In vitro Transcription of 7SK RNA .........................................................72 Hybridization and Primer Extension Reactions ......................................72
Results .............................................................................................................73 Phosphorylation, Dephosphorylation, and Acetylation of the 7SK snRNP does not Result in P-TEFb Release .............................................73 DSIF, Gdown1, DRB and Flavopiridol do not Cause P-TEFb Release Directly .......................................................................................75 HIV Tat and Brd4 Can Release P-TEFb Directly from the 7SK snRNP ......................................................................................................76 A Conformational Change Occurs in 7SK snRNA After P-TEFb Release Preventing the Binding of HEXIM1 ..........................................79
CHAPTER 5 SUMMARY AND FUTURE DIRECTIONS ............................................116 LARP7 Stabilizes 7SK snRNA in Human Cells ..........................................117 Conservation and Regulation of P-TEFb by the 7SK snRNP in Drosophila .....................................................................................................118 Regulated Release of P-TEFb from the 7SK snRNP for Viral and Cellular Gain .................................................................................................119
Figure 1: Model of transcription elongation control and P-TEFb regulation ....................12
Figure 2: Expression and purification of human LARP7 for antibody generation ............26
Figure 3: LARP7 co-sediments with the 7SK snRNP before and after the release ofP-TEFb and HEXIM..............................................................................................28
Figure 4: 7SK snRNA exhibits the same glycerol gradient sedimentation pattern as LARP7 ......................................................................................................................30
Figure 5:LARP7 is a stable component of the 7SK snRNP ...............................................32
Figure 6: Knockdown of LARP7 results in a relative increase of free P-TEFb but an overall decrease of total P-TEFb ..............................................................................34
Figure 7: LARP7 and a model of current mechanism of P-TEFb release .........................36
Figure 8: Schematic diagram of LARP7 conservation in eukaryotes ................................52
Figure 9: Expression and purification of dLARP7 for affinity purified antibody generation..................................................................................................................54
Figure 10: dLARP7, dHEXIM, and CyclinT co-sediment and co-immunoprecipitate in an RNase sensitive complex ..................................................56
Figure 11: d7SK RNA is conserved in Drosophila and immunoprecipitates with dLARP7 ....................................................................................................................58
Figure 12: d7SK co-immunoprecipitates with dHEXIM, dLARP7, and CyclinT .............60
Figure 13: A comparison of the human and Drosophila 7SK snRNP ...............................62
Figure 14: Phosphorylation, dephosphorylation, and acetylation do not release P-TEFb .........................................................................................................................88
Figure 15: Negative elongation factors, Myc, and ATP analogs do not cause release of P-TEFb .................................................................................................................90
Figure 16: Schematic of mutants and expression in E. coli ...............................................92
Figure 19: The RNA binding domain of Tat is not required for P-TEFb release ..............98
Figure 20: Brd4 can extract P-TEFb directly from the 7SK snRNP ................................100
Figure 21: Summary and quantification of the Brd4 release data ...................................102
Figure 22: Schematic of TAR and 7SK RNA secondary structure .................................104
viii
Figure 23: CMCT modification and primer extension ....................................................106
Figure 24: Release of P-TEFb by flavopiridol causes a conformational change in 7SK .........................................................................................................................108
Figure 25: Tat release of P-TEFb from the 7SK snRNP causes a conformational change in 7SK and results in HEXIM release from the complex ...........................110
Figure 26: Model of P-TEFb release from the 7SK snRNP ............................................112
Figure 27: Model of P-TEFb release by Brd4 and Tat ....................................................114
CPSF Cleavage-Polyadenylation Specificity Factor CTD Carboxy Terminal Domain Ctk1 CTD Kinase 1 CtsF Cleavage Stimulation Factor d7SK Drosophila La Related Protein 7 dHEXIM Drosophila Hexamethylene Bisacetamide Induced 1 dLARP7 Drosophila La Related Protein 7 DMSO Dimethyl sulfoxide DNA Deoxyribonucleic Acid DRB 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole DSIF DRB Sensitivity Inducing Factor EBNA2 Epstein-Barr Virus EBV Epstein-Barr Virus EtBr Ethidium Bromide FPLC Fast Protein Liquid Chromatography HEXIM1 Hexamethylene Bisacetamide Induced 1 HEXIM2 Hexamethylene Bisacetamide Induced 2 HIV Human Immunodeficiency Virus HMBA Hexamethylene Bisacetamide hnRNP Heterogeneous Nuclear Ribonucleoprotein HSP70 Heat Shock Protein 70 HSV Herpes Simplex Virus HTLV Human T-lymphotropic virus IL-6 Interleukin-6 IP Immunoprecipitation
x
LARP7 La Related Protein 7 LTR Long Terminal Repeat m7G 7-methyl-guanosine MePCE Methyl-phosphate Capping Enzyme mRNA Messenger RNA MXC Multi Sex Combs myc Myelocytomatosis Viral Oncogene MyoD Myoblast Determination Protein NE Nuclear Extract NELF Negative ELongation Factor NF-κB Nuclear Factor kappa-light-chain-enhancer of Activated B cells N-TEF Negative Transcription Elongation Factor p65/RELA Reticuloendotheliosis Viral Oncogene PAF Polymerase II-Associated Factor Complex PI3K Phosphoinositide 3-kinase PKC Protein Kinase C poly(A) poly adenosine PP1α Protein Phosphatase 1 alpha P-TEF Positive Transcription Elongation Factor RNA Ribonucleic Acid RNAi RNA interference RNAPII RNA Polymerase II RNAPIII RNA polymerase III RPB1 RNA Polymerase core protein 1 RPBII RNA Polymerase core protein 2 RRM1 RNA Recognition Motif 1 RRM3 RNA Recognition Motif 1 Ser Serine siRNA Small Interfering RNA snRNA Small Nuclear RNA snRNP Small Nuclear Ribonucleoprotein TAF TBP Associated Factor TAK Tat Associated Kinase TAP-tagging Tandem Affinity Purification tagging TAR Trans-activation Response Element Tat Trans-Activator of Transcription TatC Tat CyclinT1 Binding Mutant TatR Tat RNA Binding Mutant Tax Transcriptional activator TBP TATA Binding Protein TFIIA Transcription Factor for polymerase II A
xi
TFIIB Transcription Factor for polymerase II B TFIID Transcription Factor for polymerase II D TFIIE Transcription Factor for polymerase II E TFIIF Transcription Factor for polymerase II F TFIIH Transcription Factor for polymerase II H Thr Threonine TNF Tumor Necrosis Factor TSS Transcription Start Site TTF2 Transcription Termination Factor 2 UCSC University of California Santa Cruz UTR Untranslated Region UV Ultraviolet VP16 Viral Protein 16 WCE Whole Cell Extract
1
CHAPTER 1
INTRODUCTION
The amount of observable diversity in our world is astounding. From the people
we interact with on a daily basis to the model organisms that we use in the lab, we are
surrounded by diversity. One of the central goals of biology has been to understand how
this diversity is produced. In 1869, Friedrich Miescher first described nuclein which he
isolated from the nuclei of white blood cells. Sixty years later, in 1928, Frederick
Griffith discovered the transformative properties of mixing killed virulent bacteria with
non-virulent bacteria, showing that virulence could be transferred via the genetic material
to innocuous strains of bacteria. Although disputed for years by Linus Pauling and others
who favored protein as the genetic material, Avery, MacLeod, and McCarty showed that
deoxyribonucleic acid (DNA) conferred virulence in Griffith’s 1928 work. The
discovery of the genetic material, its crystal structure, and the development of the central
dogma provided the basis for understanding how the genetic material contributes to
diversity.
Though scientists knew that DNA, its replication or copying, and its conversion
into ribonucleic acid (RNA) were regulated processes, the importance of this regulation
in controlling diversity could not be fully appreciated until the sequencing of multiple
genomes. In September of 2005, Nature magazine published the comparison of the
Human and Chimpanzee genome sequences. It was found that although chimps and
humans differ significantly in appearance, they only differ on the genetic level by 1.23%
(Consortium, 2005). This fact shed significant light on the importance of the regulation
of gene expression and put its associated fields in the spotlight. Since these early
discoveries, chromatin packaging, epigenetics, and transcription regulation have been
invaluable in helping to explain how genetically similar animals can appear so distinctly
different.
2
The molecular biology and biochemistry behind transcription, or the conversion
of the DNA message into RNA, has been studied for decades. The focus of many of the
early studies in this field has been on initiation, or how the polymerase is loaded onto the
promoter. Research on the important factors controlling transcription elongation and
termination has lagged behind as a result. More recently, the discovery that the majority
of actively transcribed genes have RNA polymerase II (RNAPII) poised just downstream
of their promoters has added a level of complexity to gene expression that was previously
underappreciated by many in the transcription field (Guenther et al., 2007; Muse et al.,
2007; Zeitlinger et al., 2007). RNAPII elongation control is now recognized as an
important regulatory step in gene expression and not as an artifact of in vitro transcription
assays or a special case at the HSP70 promoter (Rasmussen and Lis, 1993).
The RNA Polymerases
There are three major RNA polymerases in the cell and each performs specific
functions. RNA polymerase I localizes to the nucleolus of the nucleus and transcribes the
28S, 5.8S, and 18S ribosomal RNAs required for the assembly of the ribosome. RNA
polymerase III is responsible for the transcription of the transfer RNAs, 5S ribosomal
RNA, many of the small nuclear RNAs (snRNA) including 7SK, and the splicing RNAs.
The focus of this thesis will be on the steps that lead to the regulation of RNA
polymerase II, which is responsible for the transcription of microRNAs, some snRNAs,
and protein coding mRNAs.
RNAPII is a 550kD protein complex composed of 12 subunits. The two largest
subunits of eukaryotic RNAPII (RPB1 and RPB2) make up the bulk of the protein
complex, and RPB1 contains an essential Carboxyl Terminal Domain (CTD) that is
critical for regulation and cell viability (Darst et al., 1991; Meredith et al., 1996).
RNAPII catalyzes the production of mRNA through three distinct phases: initiation,
3
elongation, and termination. Each phase of transcription is thought to be a highly
regulated process requiring the concerted efforts of multiple protein complexes.
Transcription Initiation
Localizing RNAPII to gene promoters is a complicated process. Its recruitment is
dependent on a variety of factors. Cis acting elements in the DNA sequence are
important for recruiting transcription factors (trans acting factors) to enhancer elements
(Szutorisz et al., 2005). A well studied studied cis acting element is the TATA box that
is recognized by TATA binding protein (TBP) and is located approximately 25 base pairs
(bp) upstream of the transcription start site (TSS) (Comai et al., 1992; Cormack and
Struhl, 1992; Killeen et al., 1992). Other enhancer sequences can be found very far away
from TSSs and are important for opening chromatin, recruiting activator proteins, and
regulating initiation complex formation (Woychik and Hampsey, 2002).
TBP and TBP associated factors (TAFs) make up Transcription Factor for
polymerase II D (TFIID) which is the first protein complex involved in forming the pre-
initiation complex that recruits RNAPII to the promoter. TFIID is then joined by a host
of transcription factors including TFIIA and TFIIB which stabilize the interaction of
TFIID with the TATA box (Conaway and Conaway, 1993). TFIIF then recruits RNAPII
to this growing complex. TFIIE and TFIIH join the complex at the same time. TFIIH
contains the cyclin dependent kinase (CDK), CDK7/CyclinH, and a DNA helicase
required for the synthesis of the first 8-12 nucleotides (Conaway and Conaway, 1993).
CDK7 plays an important role in regulating RNAPII by phosphorylating Serine 5 of the
heptapeptide repeat YSPTSPS of the RNAPII CTD (Roeder, 1996). This results in
promoter clearance, the synthesis of the first 30-50 bases of the transcript, and RNA
capping by the capping enzyme which adds a protective 7-methylguanosime (m7G) cap to
the nascent RNA.
4
RNAPII Transcription Elongation Control
After promoter clearance, RNAPII comes under the control of Negative
Transcription Elongation Factors (N-TEFs) that cause the promoter proximal pausing of
RNAPII. The founding member of the N-TEFs is DRB Sensitivity Inducing Factor
(DSIF) which was first discovered while studying the effects of the ATP analog 5,6-
Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) on RNAPII. It had been known for
decade that treatment of cells with DRB resulted in the stalling of RNAPII in in vitro
transcription reactions with crude extracts, but this drug had no effect on purified
RNAPII (Chodosh et al., 1989). The protein responsible for this activity, DSIF, was
finally found by fractionating HeLa nuclear extract and then adding those fractions to
purified RNAPII until a DRB sensitive fraction was discovered (Wada et al., 1998a).
DSIF is composed of spt4 and spt5 and has functional homologues through yeasts
(Peterlin and Price, 2006). It was not long after that a second factor, Negative
ELongation Factor (NELF), involved in promoter proximal pausing was discovered and
later shown to be required for the negative effects of DSIF (Renner et al., 2001;
Yamaguchi et al., 1999). Promoter proximal pausing provides the cell with a significant
point for gene expression regulation (Figure 1A).
The negative effects of DSIF and NELF can be reversed by the Positive
Transcription Factor (P-TEF) P-TEFb that is a kinase composed of one of three cyclins
(cyclin T1, cyclin T2, or cyclin K) and one of two cyclin dependent kinases (CDK9 or a
larger isoform CDK955). P-TEFb, which was discovered by adding fractionated
Drosophila Kc cell lysates back to purified elongation complexes to reconstitute DRB
sensitive transcription, alleviates the stall on the paused polymerase and phosphorylates
the CTD of RNAPII on Serine 2 of the heptapeptide repeat (Marshall and Price, 1995).
Phosphorylation of the NELF-e subunit of NELF by P-TEFb causes NELF to leave the
elongation complex and phosphorylation of the spt5 subunit of DSIF by P-TEFb turns
DSIF from a negative factor into a positive factor (Ping and Rana, 2001; Wada et al.,
5
1998b) (Figure 1A). After P-TEFb function, TFIIF rebinds the freed polymerase to
stimulate transcription elongation (Cheng and Price, 2007).
As RNAPII elongates and synthesizes RNA, it produces a number of distinct
regions. The 5′ untranslated region (UTR) is a stretch of RNA between the cap structure
and the translation start site. The polymerase continues through the coding sequence
(CDS) that is defined by the translation start and stop sites. The polymerase continues to
synthesize RNA and produces the 3′ UTR. Both the 5′ and 3′ UTRs of messenger RNA
(mRNA) have been shown to be important for the regulation of translation as targets of
both proteins and microRNAs that modulate protein translation. Finally, the polymerase
transcribes a poly adenylation signal that results in the addition of a poly(A) tail.
Transcription Termination and 3′ End Formation
Transcription termination is the process in which the RNAPII releases both the
polymerase and the RNA transcript from the DNA template. This process is not well
understood in eukaryotes. An ATP dependent SWI/SNF family protein, transcription
termination factor 2 (TTF2) has been described and is involved in terminating
polymerases before entry into mitosis (Jiang et al., 2004). The role of TTF2 in general
transcription termination has not been explored further. There is also evidence that
RNAPII termination in yeast may be caused by an RNA helicase, Sen1p and the
nucleases Rat1p and Rnt1p, that scan the RNA until they encounter the polymerase and
cause it to dissociate from the DNA template; however, this torpedo model of termination
is controversial (Kawauchi et al., 2008).
Although the exact process of RNAPII termination is not well understood, it is
thought to be linked to 3′ end formation (Bentley, 1999; Neugebauer, 2002). Most
protein coding mRNA contains a poly(A) tail that is important for mRNA stability,
promotes nuclear export and is required for efficient translation of the message by the
ribosome. 3′ end formation is catalyzed by the cleavage-polyadenylation specificity
6
factor (CPSF) that scans the nascent RNA for its binding recognition sequence,
AAUAAA. This results in the recruitment of cleavage stimulation factor (CstF),
cleavage factors I and II, and poly(A) polymerase which cleave the nascent transcript
near the CPSF binding recognition sequence and catalyze the addition of the poly(A) tail
(Zorio and Bentley, 2004). The fate of the polymerase after this cleavage is not known,
but it is assumed to be terminated.
Additionally, termination in the classical prokaryotic sense may not occur in
eukaryotes. A new theory has emerged that suggests the 5′ and 3′ ends of the DNA
coding sequence may form loops, much like the loops formed by mRNA to increase
ribosome translation efficiency (Ansari and Hampsey, 2005; O'Sullivan et al., 2004;
Perkins et al., 2008). This would provide an elegant mechanism for the reloading of
polymerase at the TSS.
Regulation of P-TEFb by the 7SK snRNP
Controlling the fate of RNAPII is extremely important for gene regulation. This
is highlighted exquisitely in cancers in which many oncogenes lead to the deregulation of
transcription by removing repressors and taking advantage of activators to promote
cellular proliferation. The recent finding that a large number of actively transcribed
genes have poised polymerases (Guenther et al., 2007; Muse et al., 2007; Zeitlinger et
al., 2007) underscores the importance of tightly controlling the only known factor
implicated in releasing the polymerase from this poised state, P-TEFb.
P-TEFb is present in two forms within the nucleus: the small active form that is
involved in phosphorylating the CTD of RNAPII, and the large inactive form that is
comprised of P-TEFb, HEXIM1 or HEXIM2, and the small nuclear RNA (snRNA) 7SK
(Byers et al., 2005; Yik et al., 2003) (Figure 1B). HEXIM, or hexamethylene-bis-
acetamide-inducible, was first discovered as a protein whose expression increased
dramatically after treatment of cells with HMBA (Kusuhara, 1999), but later it was
7
shown to be the P-TEFb inhibitory protein (Yik et al., 2003). Both HEXIM and the
CyclinT1 subunit of P-TEFb are capable of binding 7SK; however, binding of P-TEFb to
7SK without HEXIM is not sufficient for inhibition in vitro (Michels et al., 2003; Yik et
al., 2003). It is believed that HEXIM and 7SK form an inhibitory complex with P-TEFb
to prevent aberrant activation of RNAPII transcription and to serve as a readily accessible
pool that can be activated during times of stress (Nguyen et al., 2001). After P-TEFb
release, HEXIM leaves the complex and the RNA is protected from degradation by the
binding of hnRNP proteins (Krueger et al., 2008; Van Herreweghe et al., 2007) (Figure
1B).
How regulated release of P-TEFb from the 7SK snRNP occurs is not understood.
Phosphorylation of threonine 186 on CDK9 is required for P-TEFb to complex with 7SK
(Chen et al., 2004), and while dephosphorylation would likely lead to the release of P-
TEFb from the large complex (Chen et al., 2008), it would be catalytically inactive. It
has also been suggested that activation of the PI3K/Akt pathway by HMBA treatment of
cells results in the release of P-TEFb from the 7SK snRNP through the phosphorylation
of HEXIM1(Barboric et al., 2007; Contreras et al., 2007). Finally, acetylation of P-TEFb
also appears to be a hallmark of free active P-TEFb and may play an important role in
release (Cho et al., 2009). The current body of literature provides evidence that post-
translational modifications are important for releasing P-TEFb from the 7SK snRNP to
activate transcription; however, specific extraction of P-TEFb from this complex is also a
possibility.
Gene Specific Regulation of Transcription by P-TEFb
The discovery of P-TEFb has been significant for many reasons. RNAPII
elongation control has since emerged as an important stage in regulating gene expression.
P-TEFb is likely to be intricately regulated during development, stress responses, and
disease states (Peterlin and Price, 2006). Although P-TEFb appears to be required as a
8
general transcription factor, cases of gene specific regulation by P-TEFb are known. The
first of these was the discovery that P-TEFb was the HIV-Tat associated kinase (TAK)
(Zhu et al., 1997). Human immunodeficiency virus (HIV) expresses a small 15 kD
protein Trans-Activator of Transcription (Tat) which is required for HIV replication
(Frankel, 1992; Laspia et al., 1989). Tat expression and recruitment of P-TEFb to the
HIV long terminal repeat (LTR) are required for the vast increase in HIV RNA that is
seen during HIV virulency. Tat binds specifically to CyclinT1 of P-TEFb through a zinc
binding domain and recruits P-TEFb to the HIV LTR by binding to a viral RNA stem
loop, the Trans-Activation Response element (TAR) (Dingwall et al., 1990; Dingwall et
al., 1989). HIV, however, is not the only virus that takes advantage of P-TEFb. Human
T-lymphotropic virus (HTLV) has been shown to encode a transcriptional activator (Tax)
that functions similarly to Tat by binding to a region in CyclinT1 of P-TEFb and
recruiting it to the HTLV viral promoter (Rosenblatt et al., 1988; Zhou et al., 2006).
Gene specific regulation is not limited to viral high-jacking of P-TEFb. Other
endogenous transcription activators have been shown to associate with and recruit P-
TEFb specifically to RNAPII promoters. These include the p65/RELA subunit of nuclear
factor kappa-light-chain-enhancer of activated B cells (NF-κB), class II major
histocompatibility complex transactivator (CIITA), the transcription factor Myc, the
myogenic regulatory factor MyoD, and a number of nuclear receptors including the
androgen receptor (Lee and Chang, 2003; Lee et al., 2001), and estrogen receptor
(Wittmann et al., 2005). All of these interactions act to recruit P-TEFb to sites of
transcription and activate RNAPII. A bromodomain protein, Brd4, has also been
discovered that binds to CyclinT1 and recruits P-TEFb to sites of acetylated and
potentially active chromatin (Jang et al., 2005; Yang et al., 2005). It seems likely that
these activator proteins serve as the link between transcription initiation and elongation.
Their role as P-TEFb recruiters is important; however, a more general mechanism for
9
P-TEFb release and recruitment outside of this small subset of special cases is still
elusive.
P-TEFb Regulation and Associated Diseases
The association of P-TEFb with disease is not surprising given its tissue
expression profile and connections with cellular differentiation. P-TEFb is highly
expressed in terminally differentiated tissues such as brain and muscle, but is also found
in proliferating cells (Bagella et al., 1998). It has been shown to be an important factor in
regulating the differentiation of neuronal, muscle and lymphocytic cells (Marshall and
Grana, 2006). The role of P-TEFb during embryonic development is also likely to be
important and highly regulated. Receptor specific regulation of P-TEFb has also been
described for tumor necrosis factor (TNF) and interleukin-6 (IL-6) implicating P-TEFb as
a central player in inflammation and cell growth and survival (Brasier, 2008; Falco et al.,
2002; Hou et al., 2007; Shan et al., 2005).
Cancerous transformation of cells is typically accompanied by deregulation and
over-expression of anti-apoptotic and pro-survival proteins. The use of flavopiridol, a
potent inhibitor of CDK9, in phase II clinical trials is an indication of the importance of
P-TEFb in both transcription and cancer. Inhibition of P-TEFb is associated with
hypophosphorylation of the CTD of RNAPII and p53 dependent apoptosis in chronic
lymphocytic leukemia (CLL) (Alvi et al., 2005) and other leukemia cells (Gao et al.,
2006). Inhibition of P-TEFb will be relevant for a wide variety of cancers because P-
TEFb has been shown to be associated with both Myc and NF-κB (Shapiro, 2006).
Finally, P-TEFb is implicated in both prostate and breast cancer due to its association
with the androgen receptor (Lee and Chang, 2003; Lee et al., 2001) and estrogen receptor
(Wittmann et al., 2005).
The importance of P-TEFb in HIV and HTLV was discussed earlier in this
chapter, but it is also significant to note that other viruses take advantage of P-TEFb.
10
EBNA2 of Epstein-Barr Virus (EBV) has been shown to be dependent on the CTD kinase
activity of P-TEFb. Both expression of a dominant negative version of CDK9 or
treatment of EBV infected cells with DRB results inhibition of EBV viral gene
expression (Bark-Jones et al., 2006). Additionally, VP16 of herpes simplex virus has
been shown to interact directly with CyclinT1 and may be important for recruiting P-
TEFb to herpes virus promoters (Kurosu and Peterlin, 2004).
Myocardial infarction, or heart attack, is a leading cause of death in developed
countries with 1 in 5 at risk of suffering a heart attack in the United States alone. Since
cardiac myocytes are terminally differentiated, they do not proliferate after enduring
stress, they become larger or hypotrophic. The molecular hallmarks of cardiac
hypertrophy include an increase in cell size and a dramatic increase in both RNAPII
transcription and cellular mRNA content. As a result, it seems obvious that P-TEFb
would be closely associated with cardiac hypertrophy (Sano et al., 2002; Sano and
Schneider, 2004; Sano et al., 2004). Activation of P-TEFb using anti-sense 7SK in
cardiac cell cultures or heart specific over expression of P-TEFb in mice recapitulated the
effects seen in mice in which cardiac infarction was induced (Sano and Schneider, 2004).
Furthermore, the mouse cardiac lineage protein-1 (CLP-1) that was shown to be
important for regulating cardiac hypertrophy in mice actually turned out to be the mouse
homologue of human HEXIM1 (Huang et al., 2002). Mice with a CLP-1/HEXIM1
knockout die early in fetal development and show all of the genetic and physical
characterisitcs of cardiac hypertrophy (Huang et al., 2004).
The association of P-TEFb with at least two major causes of death in the
developed world (heart disease and cancer) and one in the developing world (HIV), make
it a medically relevant protein complex. Further study of P-TEFb, its activation, and
regulation of transcription are essential for understanding the pathogenesis these diseases.
11
Focus of the Thesis
The goal of the research presented in this thesis is to better understand how the
7SK snRNP regulates P-TEFb and gene expression. Chapter 2 describes the discovery
and characterization of the 7SK stability protein, La related protein 7 (LARP7). These
data show that LARP7 is one of the only proteins that remains associated with 7SK after
P-TEFb and HEXIM1 are released. Loss of LARP7 also results in an increase in free P-
TEFb and a significant reduction in the amount of 7SK in the cell. These results indicate
that the maintenance of the 7SK snRNP is important for transcription regulation and
survival.
In Chapter 3, the conservation of the 7SK snRNP is explored by characterizing
the Drosophila melanogaster homologues of LARP7 and 7SK. These studies show that
the system of P-TEFb regulation is similar in flies and this model system may be useful
in characterizing the role of P-TEFb in development.
In Chapter 4, factors and modifications involved in releasing P-TEFb are
explored. Using the LARP7 antibody developed for the studies conducted in Chapter 2,
an assay was developed for discovering proteins that can bind to and release P-TEFb
from the 7SK snRNP. This assay shows that post-translational modifications of P-TEFb
including phosphorylation of HEXIM and acetylation or dephosphorylation of P-TEFb do
not cause P-TEFb release directly. However, addition of HIV Tat, or the C-terminal
region of Brd4 are capable of extracting P-TEFb in this in vitro assay. Most importantly,
the release of P-TEFb is followed by a conformational change in 7SK RNA that prevents
the continued binding of HEXIM1 to the complex.
Finally, in Chapter 5 a summary of the significant findings is presented and future
directions are discussed.
12
Figure 1: Model of transcription elongation control and P-TEFb regulation
A) A model of transcription elongation highlighting both abortive elongation and
productive transcription elongation. After the polymerase initiates and is phosphorylated
on Ser5 by TFIIH it comes under the negative regulation of DSIF and NELF. If this
stalled polymerase is not released, transcription aborts and produces short transcripts. If
the kinase P-TEFb acts on the stalled polymerase, it phosphorylates Ser2 of the CTD, the
spt5 subunit of DSIF, and the NELF-e subunit of NELF. This results in the conversion of
DSIF from a negative factor to a positive one and the loss of NELF from the complex.
The polymerase is then released from the pause and enter productive transcription
elongation. B) P-TEFb is regulated in the cell by a cycle of inhibition by and release
from a small ribonuceloprotein complex in the nucleus. This complex is composed of the
small nuclear RNA 7SK, the major inhibitory protein HEXIM which mediates the
interaction of P-TEFb with the RNP, the stability protein LARP7, and finally the methyl
phosphate capping enzyme which adds a methyl cap to 7SK RNA and remains bound to
the complex to provide further stability. After P-TEFb is released from the 7SK snRNP,
HEXIM leaves the complex and the RNA is then bound by a variety of hnRNP proteins.
13
14
CHAPTER 2
LARP7, A NOVEL COMPONENT OF THE 7SK SNRNP STABILIZES
7SK SNRNA IN HUMAN CELLS
Regulated inhibition of P-TEFb is important for control of RNAPII transcription
elongation. P-TEFb exists in two distinct protein complexes in the cell: one that is
composed of free active CyclinT1 and CDK9 and the other in which these proteins are
bound to and inhibited by the 7SK snRNP. The goal of the research presented in this
chapter was to discover new proteins that were bound to and potentially regulate P-TEFb
release. Through a collaboration with Benoit Coulombe (Institut de recherches cliniques
de Montréal), the La Related Protein 7 (LARP7) was discovered as component of the
7SK snRNP. To confirm and further characterize this interaction, a LARP7 antibody was
developed in sheep and analyses were performed to determine the association and role of
LARP7 as a component of the 7SK snRNP. It was discovered that LARP7 co-sediments
with the 7SK snRNP and remains associated with this complex after P-TEFb release.
Co-immunoprecipitation experiments showed that LARP7 is associated with all of the
known components of the 7SK snRNP and LARP7 mediates the interaction with this
complex by binding directly to 7SK. Loss of LARP7 through siRNA knockdown
resulted in a significant decrease in 7SK snRNA and P-TEFb highlighting its importance
in maintaining the stability of the 7SK snRNP.
Introduction
P-TEFb performs an essential function in regulating the transcription of cellular
genes. Its activity is controlled by a small nuclear ribonucleoprotein complex composed
of a 332bp small nuclear RNA, 7SK (Nguyen et al., 2001; Yang et al., 2001), and the
RNA binding proteins HEXIM1 and/or HEXIM2 (Byers et al., 2005; Yik et al., 2005).
HEXIM alone is not sufficient to inhibit P-TEFb and requires RNA to cause a
conformational change in HEXIM to open up its P-TEFb binding pocket (Li et al., 2007).
15
Prior to 2007, the only known components of the 7SK snRNP were 7SK, HEXIM,
and P-TEFb; however, there was significant evidence that other proteins were likely
involved in binding to this complex. In the original classification of 7SK, Wassarman
and Steitz showed that the 7SK snRNP contained at least 5 proteins ranging in size from
40 – 120kD. We can speculate that CyclinT1 (~120kD), CDK9 (~40kD), and HEXIM1
(~60kD) account for 3 of these proteins (Wassarman and Steitz, 1991). To identify the
other proteins bound in this complex, we entered into a collaboration with Benoit
Coulombe who was conducting experiments to determine the transcription and RNA
processing proteome. He accomplished this using tandem affinity purification tagging
(TAP-tagging) of proteins coupled with tandem mass spectrometry to determine protein
interaction networks. The first screen discovered a number of very interesting proteins.
This initial experiment identified a methyl transferase that was tightly associated with
CDK9, CyclinT1, and HEXIM (Jeronimo et al., 2007). It was originally thought that this
methyl transferase would be important for modifying histones, but it was later discovered
that it added the special mono-methyl-guanosine cap to the γ-phosphate of 7SK RNA.
This factor was renamed the methyl phosphate capping enzyme because of its capping
activity (MePCE) (Jeronimo et al., 2007). This screen also identified a La related
protein, LARP7, associated with P-TEFb and HEXIM. Considering the fact that 7SK
RNA is the product of RNAPIII, it seemed fitting that a La related protein might be
helpful in stabilizing the 7SK snRNP and protecting the RNA from decay.
Autoantigen La was first identified as a highly expressed RNA binding protein
(Wolin and Cedervall, 2002). Human La protein is involved in the stabilization and
maturation of many RNAPIII transcripts (Rinke and Steitz, 1982; Rinke and Steitz,
1985). The primary function of La is to bind to the 3′ UUU-OH of RNAPIII RNAs after
transcription termination and protect them from degradation by exonucleases (Reddy et
al., 1983; Saito et al., 1994; Stefano, 1984). Modification of the 3′ UUU-OH results in
the release of La from the transcript (Stefano, 1984). La is also known to bind to the 5′
16
end of newly synthesized RNAPIII RNAs; however, removal of the triphospahte or the
addition of a methyl group decreases the affinity of La for these transcripts
(Bhattacharya et al., 2002; Fan et al., 1998; Intine et al., 2000; Maraia and Intine, 2001).
Which La domains specifically bind to each of these structures is not known, but it is
thought that the La-motif and first RNA Recognition Motif (RRM) are responsible
(Goodier et al., 1997; Ohndorf et al., 2001). La has also been credited with aiding in
nuclear retention of snRNAs and their assembly into functional RNP complexes (Boelens
et al., 1995; Grimm et al., 1997; Pannone et al., 1998; Simons et al., 1996; Xue et al.,
2000). The diverse roles that La plays in RNAPIII RNA maturation are intriguing when
considering how LARP7 may be involved in protecting 7SK or regulating P-TEFb
release from the 7SK snRNP.
Materials and Methods
Generation and Affinity Purification of LARP7 Antibodies
Human LARP7 was cloned into pET21a (Novagene) from MGC clone 87333
with a C-terminal histidine tag. The clone was transformed into Escherichia coli BL21
CodonPlus (DE3) RILX cells (Stratagene). Cells were grown to an OD of .500 and then
induced overnight at 18oC with 0.1mM IPTG. Purification was carried out by sonicating
the cells, spinning out any large insoluble material at 200 x g for 45 minutes in a
Beckmann Ultracentrifuge, and binding the expressed protein to Ni-NTA resin. The
protein eluted from the nickel resin was then further purified by fast protein liquid
chromatography (FPLC) over a mono S column. One fraction from the Mono S elution
which contained a single 38kD C-terminal LARP7 proteolysis product was used as an
antigen to generate sheep antibodies (Elmira Biologicals). LARP7 antibodies were then
affinity purified by covalently attaching LARP7 antigen to Actigel ALD beads
(Sterogene) according to the manufacturer’s recommendation for antibody affinity
17
purification. Sheep serum was bound to this column and then affinity purified antibodies
were eluted with Tris-glycine buffer.
Glycerol Gradient Sedimentation Analysis
HeLa cells were grown to 90% confluence in T-150 flasks. Half of the flasks
were then treated for 1 h with 500 nM flavopiridol while the other half were mock treat
with 0.004% DMSO carrier. Cells were then harvested, washed, lysed with a buffer
consisting of 10 mM HEPES, 150 mM NaCl, 2 mM MgCl2, 10 mM KCl, 0.5% NP-40,
0.5 mM EDTA, 1 mM DTT, 0.1% PMSF, 1U/mL Roche EDTA free complete protease
inhibitor cocktail, and 10U/mL RNaseOut (Invitrogen) and subjected to glycerol gradient
fractionation over a 5%-45% glycerol range. This was done by overnight (16 h)
ultracentrifugation at 200xg in a Beckmann Ultracentrifuge. Gradients were then
fractionated in to 16 fractions and resolved by 9% SDS-PAGE followed by transfer to
nitrocellulose membranes.
Western Analysis
After transfer to nitrocellulose, the membranes were cut and incubated in 0.1%
tween PBS and 3% milk overnight at 4oC with the appropriate primary antibodies.
Membranes were then washed 3 times in 0.1% tween PBS and incubated with
horseradish peroxidase-conjugated secondary antibody (Sigma). The membranes were
treated with Super Signal Dura West Extended Duration Substrate (Pierce) and imaged
using a cooled charge-coupled camera (UVP).
The antibodies used for western analysis were: sheep anti-cyclin T1, rabbit anti-
CDK9 (sc-8338; Santa Cruz Biotechnology), affinity-purified sheep anti-HEXIM1, rabbit
anti-MEPCE, and affinity-purified sheep anti-LARP7.
18
Immunoprecipitation
Fractions 8-11 of control and flavopiridol-treated glycerol gradients were pooled
because they contain the majority LARP7 and the P-TEFb containing 7SK snRNP.
Affinity purified LARP7 antibodies were covalently attached to actigel ALD beads
(Sterogene) according to the manufacturer’s instructions for immunoprecipitation. The
pooled glycerol gradients were pre-cleared with beads alone at 4oC for 1 h. The flow
through was then added to the beads bound with affinity purified LARP7 antibody or to
beads alone and rotated for 1 h at 4oC. The beads were washed with IP wash buffer (10
mM HEPES, 2 mM MgCl2, 10 mM KCl, 0.1% NP-40, 0.5 mM EDTA, 150 mM NaCl.
Trizol reagent (Invitrogen) was added directly to the beads to extract immunoprecipitated
7SK according to the manufacturer’s protocol. Extracted RNA was resolved on a
Silver stain by Jeff Cooper. C) Brd4 was titrated in to the release reaction at 30, 100, 300
or 900ng for 15 minutes or the reactions contained 200ng of Brd4 for 3, 10, or 30
minutes. D) Same as in C except the Brd4 mutant missing helical domain 3 was used for
the reactions.
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Figure 21: Summary and quantification of the Brd4 release data
Brd4 release was quantified from three independent experiments. Two
independent experiments were done to calculate the mean for the Brd4 helical domain 3
mutant (Brd4M). All error bars represent standard error. The y-axis is a measure of
percent of Cdk9 left in the complex. Brd4 – Purple, Brd4M - Blue
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Figure 22: Schematic of TAR and 7SK RNA secondary structure
HIV-1 TAR RNA stem loop, 35-61 of TAR RNA covering the Tat binding bulge
(AUCUG) and the CyclinT1 binding loop (CUGGG). mFold predicted, Predicted
structure of the 1-100 region of 7SK. Wassarman, Structure of the 1-100 region of 7SK
RNA described by Wassarman and Steitz (Wassarman and Steitz, 1991). The Uracil
residues in 7SK RNA are marked, orange dashed circles highlight AUCUG regions and
blue dashed circles highlight CUGGG or CUGCG regions of RNA.
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106
Figure 23: CMCT modification and primer extension
A) CMCT covalently modifies N-3 of uracil. B) Radioactively labeled primers
are hybridized with CMCT modified RNA for primer extension by reverse transcriptase.
CMCT modification (denoted by asterisks) prevents further extension of the primer by
reverse transcriptase. The extension ladder is then visualized on 12% acrylanide gels.
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108
Figure 24: Release of P-TEFb by flavopiridol causes a conformational change in 7SK
Primer extension over the 1-70 region (70-90 primer) and the 1-100 region (100-
120 primer) are shown along with a sequencing ladder showing the position of uracil in
the RNA. The mFold and Wassarman (Wassarman and Steitz, 1991) structures are
included for a comparison of sensitivities. Green asterisk marks U28 and U30 on both
structures and the extension scans, the red asterisk marks U66 and U68.
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110
Figure 25: Tat release of P-TEFb from the 7SK snRNP causes a conformational change in 7SK and results in HEXIM release from the complex
A) Western analysis of Tat release from the 7SK snRNP after co-incubation of
Tat and immunoprecipitation media. LARP7 and CDK9 were analyzed by western. I –
Input, C – Control, T – Tat treated, Bound – Bound to the beads, FT – Flow through from
the beads. B) Primer extension analysis of the 1-70 (70-90 primer) and the 1-100 (100-
120 primer) regions of 7SK RNA structure. Asterisks are the same as in Figure 21 to
refer back to the structures. C) Graphical summary of the 7SK structural change data.
Bases with single stranded character are boxed in black. Bases bound in double stranded
character are unboxed. Uracils of interest are highlighted with structural change
information. Asterisk – change in character, minus – No change in character. D) Re-
analysis of the Tat release analysis performed in Figure 13A to determine HEXIM1
release.
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Figure 26: Model of P-TEFb release from the 7SK snRNP
A model of factors and modifications that may be important for P-TEFb release
from the 7SK snRNP. These include decay of 7SK snRNA or changes to its structure,
competition or extraction of P-TEFb by specific factors such as HIV Tat and Brd4, Post-
translational modifications to P-TEFb or HEXIM. P-TEFb can be acetylated on lysine
404 by P300, but this does not lead to release directly in the in vitro assay (Cho et al.,
2009). P-TEFb can undergo dephosphorylation of its T-Loop at Threonine 186 by PP1α
(Chen et al., 2008). Phosphorylation of HEXIM has been shown to occur by Akt in the
P-TEFb binding domain of HEXIM at Threonine 270 and at Serine 278. The RNA
binding region of HEXIM can also be phosphorylated in vitro by PKC preventing the
binding to 7SK RNA and preventing the inhibition of P-TEFb (unpublished data).
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Figure 27: Model of P-TEFb release by Brd4 and Tat
A) Histones are aceylated and transcription factors are recruited to the promoter
to initiate the polymerase. Phosphorylation of the CTD by TFIIH results in the opening
of the transcription bubble and the first 30-50 bases of the transcript are transcribed until
the polymerase comes under the negative regulation of DSIF and NELF. Brd4 is
recruited to acetylated lysines and waits until the 7SK snRNP comes close to the initiated
promoter. B) The C-terminus of Brd4 binds to and extracts P-TEFb from the 7SK snRNP
and tethers it to the promoter region to phosphorylate the CTD of RNAPII on Ser2 and
also phosphorylate DSIF and NELF. C) In the case of HIV Tat, Tat binds to P-TEFb and
extracts it from HEXIM. D) Tat recruits P-TEFb to the HIV LTR by binding to the TAR
element. This results in CTD, DSIF, and NELF phosphorylation. E) These
phosphorylation events lead to productive transcription elongation. Since P-TEFb has
left the 7SK snRNP, a structural change in the RNA forces HEXIM out of the complex
and the snRNP is then bound and protected by the hnRNP proteins.
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CHAPTER 5
SUMMARY AND FUTURE DIRECTIONS
Proper expression of the genetic information is critical for normal cell function
and survival. This is highlighted exquisitely in cancer where the checks and balances that
are normally in place to ensure proper gene expression are lost and lead to unregulated
growth and proliferation. Tight control of gene expression is also required during
development where specific genetic programs are activated to produce specialized cells
and tissues. The role of transcription regulation of gene expression has been studied for
nearly half of a century; however, the majority of these studies have focused on
transcription initiation because it was believed that the regulation of polymerase loading
was the limiting step in gene expression. The discovery that polymerase complexes poise
or pause after initiation has been studied for 20 years, but many in the transcription field
believed that this pausing was a special case at specific genes or an artifact of in vitro
transcription assays. Recently, it was shown that 80% of actively transcribe genes have
paused polymerases just downstream of their promoters (Guenther et al., 2007; Muse et
al., 2007; Zeitlinger et al., 2007). Regulation of transcription elongation has since been
accepted as an important control point for gene expression. The protein responsible for
releasing the polymerase from this paused state is P-TEFb.
Poised polymerases are thought to exist to provide the cell with the ability to
rapidly respond to stress or environmental changes that require differential gene
expression. P-TEFb is a potent activator of transcription and its activity in the cell is
regulated by its sequestration in the 7SK snRNP. How P-TEFb release from this
complex is regulated has been the topic of intense research, but few questions have been
answered. The focus of the research presented in this thesis was to determine how P-
TEFb releases from the 7SK snRNP. In Chapter 2, the discovery and function of the 7SK
stability protein, LARP7, was discussed. In Chapter 3, the conservation of the 7SK
117
snRNP and its function in Drosophila melanogaster was characterized. Finally, Chapter
4 explored how both viral and cellular factors exploit P-TEFb to promote gene
expression.
LARP7 Stabilizes 7SK snRNA in Human Cells
In human cells P-TEFb is inhibited by the 7SK snRNP. LARP7 was found to be
an important stability factor that binds to and protects the RNA component of the RNP
from degradation. This is highlighted by the fact that loss of LARP7 through RNAi
knock down resulted in a significant reduction in the total amount of 7SK RNA in the
cell. Loss of LARP7 also caused a small but significant increase in the amount of free P-
TEFb ultimately resulting in the activation of a compensatory mechanism to reduce the
total amount of P-TEFb in the cell. Glycerol gradient sedimentation analysis and co-
immunoprecipitations showed that LARP7 is bound to 7SK in the snRNP regardless of
the presence of P-TEFb or HEXIM.
In the future, it would be interesting to determine how modifications to LARP7 or
7SK affect the association of LARP7 with the complex. Although LARP7 associates
with a mature RNP, its association with the RNP may be regulated. The association of
La protein with RNAPIII transcripts is regulated by post-translational modifications or
post-transcriptional modifications to its target RNAs. It would not be surprising if the
association of LARP7 with 7SK was similarly regulated. This could be done by
determining the 5′ and 3′ characteristics of 7SK RNA that are required for LARP7
association. The post-translational modification state of LARP7 before and after P-TEFb
release could also be examined to determine if specific residues are modified to promote
P-TEFb binding or release from the complex. This could easily be done by isolating the
7SK snRNP and submitting preparations for mass spectrometry analysis of LARP7.
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Conservation and Regulation of P-TEFb by the 7SK snRNP
in Drosophila
The existence and function of P-TEFb through Drosophila was known, but it was
not known whether P-TEFb was regulated similarly in Drosophila or relied on a more
primitive control mechanism. A bioinformatic screen showed that Drosophila appeared
to possess homologues of HEXIM and LARP7. The function of these proteins was then
determined by glycerol gradient analysis and co-immunprecipitations which showed that
dHEXIM, dLARP7, and the CyclinT component of P-TEFb co-sediment with one
another in an Rnase sensitive complex and that all co-immunoprecipitate with one
another. The discovery of the RNA component of the complex was confirmed and it was
shown that all three proteins are capable of associating with the RNA in vivo.
Additionally, work by a previous graduate student on this project showed that dHEXIM,
dLARP7, and CyclinT mimic their human counterparts with respect to their response
after transcription inhibition: dLAPR7 stays associated with the d7SK snRNP while
dHEXIM and CyclinT leave the complex. Characterization of this complex was an
important first step before Drosophila could be used as a model to study P-TEFb
regulation during development.
The necessity of P-TEFb and the 7SK snRNP during development is currently
being studied by our collaborators in the Matera Lab at UNC. It is assumed that P-TEFb
is an important factor in regulating embryonic development because it relieves stalled
polymerases and promotes the majority of RNAPII transcription elongation,. Its
requirement in terminal differentiation of muscle tissue and heart development has been
studied in mice, but its role in embryonic development has not been characterized. The
preliminary data from flies indicate that P-TEFb and the 7SK snRNP are present,
although differentially expressed during embryonic, larval, and adult development.
Studies characterizing the effect of complete loss of this complex will shed light on the
importance of P-TEFb during proper embryonic development.
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Regulated Release of P-TEFb from the 7SK snRNP for
Viral and Cellular Gain
P-TEFb regulation of stalled polymerases has been studied in detail; however, the
mechanisms regulating its release from the 7SK snRNP have remained elusive. It has
been known since 1997 that P-TEFb is the cellular kinase required for HIV Tat
transactivation. It was later discovered that Tat accomplishes this by binding directly to
P-TEFb and recruiting it to the HIV LTR. How Tat extracts P-TEFb from the 7SK
snRNP was not known. The data presented here show that Tat is able to extract P-TEFb
directly from the 7SK snRNP. The role of the zinc binding and RNA binding domains of
Tat were further characterized and it was shown that Tat extraction of P-TEFb is
dependent on the zinc binding region and not the RNA binding region. Like many viral
proteins, the cell has at least one protein that can mimic the actions of Tat. The
bromodomain protein Brd4 contains a P-TEFb binding domain that can also bind to P-
TEFb and extract it directly from the 7SK snRNP. Although post-translational
modifications of P-TEFb and HEXIM were thought to be important for the release of P-
TEFb from the 7SK snRNP, none of these modifications were able to do so directly.
Finally, the release of P-TEFb is followed by the concomitant release of HEXIM1 due to
a conformational change of 7SK snRNA that prevents HEXIM1 binding to ensure that
released P-TEFb arrives at its target to activate transcription.
The future directions for this research are numerous. The role of the RNA
binding domain of Tat is still not clear. It is known to bind to TAR RNA, but has greater
affinity for 7SK snRNA. The physiological relevance of this in the pathogenesis of HIV
infection needs to be explored further. Does the affinity of Tat for 7SK change after Tat
binds to P-TEFb? Do the Tat binding sites in 7SK serve any function, or is Tat binding
to 7SK a non-functional evolutionary artifact? The presence of 3 Tat binding sequences
in the first 100 bases of 7SK is not likely to be a coincidence and the conservation of
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these AUCUG repeats through Drosophila only further underscores their likely
importance in 7SK snRNP regulation of P-TEFb.
Although an endogenous protein capable of releasing P-TEFb from the 7SK
snRNP was discovered, its role in this process was not conclusively confirmed in vivo.
Knockdown of Brd4 followed by P-TEFb release signal such as flavopiridol treatment
must be conducted to determine if the loss of Brd4 prevents the rapid global release of P-
TEFb from the 7SK snRNP. Though a mechanism for release has been discovered, more
questions are raised about how P-TEFb is recruited back to the 7SK snRNP after the need
for upregulation of transcription subsides. The mechanisms regulating P-TEFb release
from Brd4 and chromatin should be explored.
The conformational change in 7SK RNA that results in the release of HEXIM1
from the 7SK snRNP is very interesting. The role that the hnRNPs or the RNA helicase
play in facilitating the maintenance or reversal of this change needs to be followed up on.
Additionally, how post-translational modifications of P-TEFb and HEXIM actually
regulate transcription should be determined. Are these modifications required for the
recruitment of P-TEFb to transcriptionally active sites, or are they only a result of P-
TEFb being in close proximity to non-specific enzymes.
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