Regulation of the Expression of Mouse Ribonucleotide Reductase Small Subunit at the Levels of Transcription and Protein Degradation
Anna Lena Chabes
Department of Medical Biochemistry and Biophysics Umeå University
Umeå 2003
Regulation of the Expression of Mouse Ribonucleotide Reductase Small Subunit at the Levels of Transcription and Protein Degradation
Copyright Anna Lena Chabes
Printed in Sweden by Solfjädern Offset AB ISBN 91-7305-468-2
Till mina föräldrar och Andrei
Table of Contents
Abstract... 2
Publication list ... 3
Part I. Introduction... 4
The cell cycle ... 5
Transcription ... 8
Protein degradation ... 12
Ribonucleotide reductase ... 15
Part II. Results... 19
Transcriptional regulation of the R2 gene... 19
Regulated protein degradation of the R2 protein ... 27
Acknowledgements... 29
References ... 30
Papers I-IV... 35
Abstract
Regulation of the Expression of Mouse Ribonucleotide Reductase Small Subunit at the Levels of Transcription and Protein Degradation
Deoxyribonucleic acid (DNA) carries all the genetic information of a cell. Ribonucleotide reductase (RNR) provides balanced pools of all four dNTPs, the building blocks of DNA.
These building blocks are needed during DNA synthesis and repair. A failure in the control of the dNTP levels and/or their relative amounts leads to cell death or genetic
abnormalities. Because of its central role in dNTP metabolism, RNR is highly regulated on multiple levels.
The active RNR enzyme consists of two non-identical subunits called proteins R1 and R2.
In mammalian cells, during an unperturbed cell cycle, the activity of RNR is highest during S and G2 phases. This is achieved by de novo synthesis of the limiting R2 protein at the onset of S phase, and by controlled degradation of the R2 protein during mitosis. This thesis deals with both the S phase-specific transcription of the mouse R2 gene, and the M phase-specific degradation of the mouse R2 protein.
Sequence comparison of the mouse R2 promoter to human and guinea pig R2 promoters revealed some conserved elements. These putative regulatory elements were tested in both in vitro and in vivo transcription assays. We demonstrated that the previously identified, NF-Y binding CCAAT box is essential for high-level expression from the R2 promoter, but not for its S phase specificity. In addition, the conserved TATA box is dispensable both for basal and S phase-specific R2 transcription as long as the first 17 basepairs of the 5’
untranslated region are present. However, if this 5’ untranslated region is absent, the TATA box is needed for correct initiation of transcription.
Focusing on the S phase specificity of the R2 gene expression, we demonstrated that the S phase-specific activity of the mouse R2 promoter is dependent on a protein-binding region located ~500 basepairs upstream of the transcription start site and an E2F binding site close to the transcription start site. Deletion of the upstream activating region results in an inactive promoter. In contrast, mutation of the E2F site leads to premature promoter activation in G1 and increased overall promoter activity. However, if the activating mutation of the E2F site is combined with mutation of the upstream activating region, the promoter becomes inactive. These results suggest that the E2F-dependent regulation is important but not sufficient for cell-cycle specific R2 transcription, and that the upstream activating region is crucial for the overall R2 promoter activity.
In our studies of the M phase-specific R2 degradation, we found that it is dependent on a KEN sequence in the N-terminus of the R2 protein, recognized by the Cdh1-APC complex.
Mutating the KEN box stabilizes the R2 protein during mitosis and G1 phase.
In summary, these studies further extend our understanding of the regulation of the limiting R2 subunit of the enzyme ribonucleotide reductase. The S phase-specific transcription of the R2 gene and the M phase-specific degradation of the R2 protein may serve as important mechanisms to protect the cell against unscheduled DNA synthesis.
Publication list
This thesis is based on the following publications, which will be referred to by their roman numerals.
I Kotova, I., Chabes, A. L., Segerman, B., Flodell, S., Thelander, L., and Björklund, S. (2001) A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH and recombinant TBP, TFIIB, TFIIE and TFIIF. Eur. J. Biochem. 268, 4527-4536
II Kotova, I., Chabes, A. L., Lobov, S., Thelander, L., and Björklund, S. (2003) Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse
ribonucleotide reductase R2 gene transcription. Eur. J. Biochem.
270, 1791-1801
III Chabes, A. L., Björklund, S., and Thelander, L. S phase-specific transcription of the mouse ribonucleotide reductase R2 gene is dependent on an upstream promoter activating region and a proximal repressive E2F binding site. Manuscript in preparation
IV Chabes, A. L., Pfleger, C. M., Kirschner, M. W., and Thelander, L.
(2003) Mouse ribonucleotide reductase R2 protein: A new target for anaphase-promoting complex-Cdh1-mediated proteolysis. Proc.
Natl. Acad. Sci. U S A. 100, 3925-3929
Part I. Introduction
All cells store their genetic information in DNA (deoxyribonucleic acid).
DNA is synthesized from four precursor building blocks - the deoxyribonucleotides (dNTPs) dATP, dCTP, dGTP and dTTP -
polymerized into a long unbranched chain. The de novo formation of all four dNTPs is catalyzed by ribonucleotide reductase (RNR) (reviewed in 1).
The dNTPs supplied by RNR are used for DNA synthesis and needed during the S phase of the cell cycle when cells duplicate their genome, and during DNA repair. The active mammalian RNR consists of two non- identical protein subunits called R1 and R2. During an unperturbed cell cycle, the limiting subunit, R2, is present only during the S and G2 phases of the cell cycle. Accordingly, an active ribonucleotide reductase enzyme is present in the S and G2 phases of the cell cycle. The restriction of RNR activity to S and G2 phases is achieved by S phase-specific transcription of the R2 gene (2) and by controlled degradation of the R2 protein during mitosis (3).
Figure 1. The activity of the enzyme ribonucleotide reductase during the cell cycle. The presence of RNR activity is regulated by de novo synthesis of the limiting R2 protein at the onset of S phase (arrow 1) and by controlled degradation of the R2 protein during mitosis (arrow 2).
Ribonucleotide Reductase activity
G1 M G1
Cell cycle progression S
Gene transcription Protein degradation
G2
1 2
This thesis presents new findings on the regulation of the cell cycle-specific transcription and protein degradation of the small RNR subunit. I first provide a brief introduction of the different players and concepts involved in these studies: the cell cycle, transcription, protein degradation, and
ribonucleotide reductase.
The Cell Cycle
A cell reproduces by duplicating its contents and dividing in two. This is done in an orderly sequence of events called the cell cycle.
Figure 2. A schematic representation of the different phases of the cell cycle with cells represented as spheres.
All the genetic information of a cell has to be accurately copied and segregated precisely into two genetically identical daughter cells. These processes define the two major phases of the cell cycle. DNA duplication occurs during S phase (S stands for synthesis), while chromosome
segregation and cell division occur during M phase (M stands for mitosis).
The M phase of the cell cycle can be divided into several stages (Figure 3).
The first one is prophase where the duplicated DNA starts to condense into
S G2
M
G1
+
well-defined chromosomes. At the end of prophase the mitotic spindle, composed of microtubules, starts to form. Next is prometaphase where the nuclear envelope breaks down allowing the spindle microtubules to interact with the chromosomes. This is followed by metaphase where the duplicated chromosomes are aligned on the mitotic spindle poised for segregation. The segregation of the chromosomes marks the beginning of anaphase. The chromosomes are pulled to the poles of the spindle and in telophase the chromosomes arrive at the poles. The nuclear envelope re-forms around each group of daughter chromosomes. The cell is pinched in two by a process called cytokinesis and the cell cycle is completed.
Figure 3. Schematic representation of the different phases of mitosis.
Apart from duplicating and dividing the DNA during the cell cycle, the cell also has to grow and double the mass of proteins and organelles in the so- called gap phases, G1 and G2. The gap phases also provide time for the cell to check that the internal and external conditions are suitable for the next phase of the cell cycle, and that the preparations for either S phase or mitosis are complete. A cell can also enter a specialized resting state called G0 (G zero).
prometa phase
metaphase anaphase telophase prophase
The cell cycle is regulated by cyclin-dependent kinases (Cdks). The activity of these kinases rises and falls as the cell progresses through the cell cycle.
The variation of Cdk-activity leads to changes in the phosphorylation state of proteins that initiate or regulate the events of the cell cycle such as DNA replication, mitosis and cytokinesis. The Cdks are only active when bound to cyclins. Similarly to the R2 protein, cyclins undergo a cycle of synthesis and degradation in each cell cycle. Consequently, the cyclic assembly and activation of the cyclin-Cdk complexes trigger the different events of the cell cycle. These activities further underscore the importance of cell cycle- specific transcription of genes, and cell cycle-specific protein degradation.
The cell cycle-specific degradation of the different cyclins and other cell cycle regulators involves the ubiquitin/26S proteasome proteolytic pathway.
Degradation via this pathway is a two-step process. The protein is first tagged by the covalent attachment of ubiquitin, a highly conserved protein of 76 amino acids. Ubiquitin-tagged proteins are subsequently degraded by a multicatalytic protease complex called the 26S proteasome. One of the ubiquitylating complexes is called the anaphase-promoting complex (APC).
The APC plays an important role in promoting anaphase by targeting an inhibitor of sister-chromatid separation, called securin, for destruction. The APC is further described in the protein degradation section below. Although the variation in cyclin levels is the primary determinant of Cdk activity during the cell cycle, Cdk activity is also regulated by other mechanisms such as phosphorylation of the cyclin-Cdk complex or binding of inhibitory proteins to the complex.
Targets of the cyclin-Cdk complexes include the retinoblastoma protein (pRB), which is phosphorylated by cyclin D-Cdk4/6 and cyclin E-Cdk2 in
the G1 phase. This phosphorylation promotes S phase entry (for review on pRB phosphorylation see 4). The retinoblastoma protein was the first identified tumor suppressor, and it is mutated in approximately one third of all human tumors (reviewed in 5). Tumors that retain wild-type pRB often carry mutations in other parts of the pRB pathway. Examples of such mutations are activating mutations of the pRB inhibitors cyclin D1 or Cdk4, or inactivating mutations of the Cdk4 inhibitor, p16. (Reviewed in 6 and 7).
pRB blocks cells in G1 by inhibiting the activity of the E2F family of
transcription factors. The E2Fs control the expression of key components of the cell cycle and DNA replication machinery. When pRB is
phosphorylated, it dissociates from E2F allowing E2F to activate its target genes. The dissociation of pRB from E2F is required for cell cycle entry (reviewed in 8 and 9). E2F activates many of the S phase-specific genes, and has been implicated in the regulation of the ribonucleotide reductase genes (10, 11, 12). The possible involvement of E2F in the regulation of the R2 gene was investigated in paper III in this thesis and will be further described below in Part II, Results.
Transcription
Copying of DNA into RNA is called transcription. Messenger RNA (mRNA) is transcribed from protein-encoding genes and is translated into proteins. RNA polymerase II catalyses transcription of all protein-encoding genes in eukaryotic cells. The polymerase is dependent on accessory proteins, called the general transcription factors (GTF). The general transcription factors TFIIB, TFIID, TFIIE, TFIIF, and TFIIH can form a multiprotein complex with RNA polymerase II that is sufficient for basal transcription in vitro (13, 14, 15). The transcription factors are binding to a region surrounding the transcription start site called a promoter. A typical
core promoter contains a conserved sequence called the TATA-box, located 20 to 30 basepairs upstream of the transcription start site. This DNA
element is recognized by the TATA-box binding protein (TBP), which is one of the subunits in the general transcription factor TFIID (16). TBP binds directly to the promoter and initiates the sequential addition of the
remaining components of the transcription initiation complex. Once the preinitiation complex is formed, the promoter DNA undergoes an ATP- dependent melting step followed by promoter clearance and synthesis of a nascent mRNA transcript. Although the general transcription factors and RNA polymerase II assemble in a stepwise mechanism in vitro, there are cases in vivo where they are brought to the promoter as a large pre-
assembled complex called the RNA polymerase II holoenzyme. In addition to the general transcription factors and the RNA polymerase II, the
holoenzyme typically contains a multi-subunit protein complex called the mediator (17).
Besides the basal transcription machinery, there are also additional
transcription factors, activators or repressors, which modulate the activity of the basal transcription machinery. This modulation is carried out by the mediator, which allows activator proteins to communicate properly with the general transcription machinery. The activators and repressors bind to enhancer or repressor elements that can be located thousands of basepairs away from the core promoter. The transcription activators usually contain a DNA binding domain (DBD) and an activation domain (AD). The main function of activators is to attract, position, and modify the general transcription factors and RNA polymerase II at the promoter so that transcription can begin.
The DNA in the eukaryotic cell is organized into discrete chromosomes each packaged into chromatin. The chromatin consists of nucleosomes: 146 basepairs of DNA wrapped around a complex of histone proteins. The activators act by binding directly, or through the mediator, to the
transcription machinery and by changing the chromatin structure around the promoter. Repressors can function by inhibiting binding of activators to the DNA, masking the activation domain of activators, blocking assembly of the general transcription machinery, or recruiting repressive chromatin remodeling and modifying complexes.
Figure 4. Schematic representation of the different players involved in transcription. The basal transcription machinery with RNA polymerase, the general transcription factors, the mediator, an activator with a DNA binding domain (DBD) and an activation domain (AD), and a nucleosome with DNA wrapped around a histone complex.
Histone deacetylase (HDAC) is a transcriptional regulator that modifies and remodels the chromatin. In the nucleosomes, each of the core histones has a long N-terminal tail, which extends out from the DNA-histone core and is
Mediator Site-specific
activator GTFs
Nuclesome
AD
RNA polymerase
subject to different types of covalent modifications. These modifications include acetylation and methylation of lysines, and phosphorylation of serines. The modifications are added and removed by chromatin remodeling factors, for example, acetyl groups are added by histone acetyl transferases (HATs) and removed by histone deacetylases. The addition of an acetyl group to the lysine of a histone tail changes the positive charge that stabilizes the interaction between the negatively charged DNA and the histones. In this way, acetylation can lead to destabilization of the chromatin structure and promote gene expression. Also, certain patterns of histone acetylation can be recognized by TFIID. In addition to the histone tail- modifying enzymes, there are ATP-dependent nucleosome-remodeling enzymes that can change the position of a nucleosome. The sliding of a nuclesome to a new position can allow a transcription factor(s) to bind to the previously blocked site and activate transcription.
One example of a family of transcription activator and repressor proteins is the aforementioned E2F family. It consists of eight genes: E2F1 to E2F6 and DP1 and DP2. The E2Fs and DPs form heterodimers with one E2F subunit and one DP subunit, where the E2F subunit provides the specific features of the heterodimer. The E2Fs can be divided into activating E2Fs and repressing E2Fs. E2F1, 2, and 3 are potent transcriptional activators.
Overexpression of E2F1-3 is sufficient to induce quiescent cells to re-enter the cell cycle. E2F4 and 5 are primarily involved in transcriptional
repression. The E2F4 and 5 are poor transcriptional activators in
overexpression assays and cannot drive quiescent cells to re-enter the cell cycle (18). Mouse embryo fibroblasts (MEFs) that are mutant for E2F4 and 5 have defects in their ability to arrest in response to growth-arrest signals (19). E2F4 and 5 are regulated by their localization. They are primarily
found in the cytoplasm, but become nuclear when bound to the pocket proteins further described below. E2F6 also acts as a transcriptional
repressor but is different from E2F4 and 5 in its structure and way of action.
E2F6 lacks the C-terminal sequences that are responsible for pocket-protein binding. Instead, the transcriptionally repressive properties of E2F6 are probably due to recruitment of non-pocket protein complexes. E2Fs are reviewed in (8) and (9).
As mentioned above, pRB acts as an inhibitor of E2F-mediated
transcriptional activation. The pRB family of proteins, also called the pocket proteins, consists of pRB, and the related p107 and p130 proteins. They all bind to and regulate the E2F proteins, but have different preferences. pRB regulates the E2F-mediated transcriptional activation by binding to the activating E2F activation domains preventing their interaction with the core transcriptional machinery. pRB can also repress transcription by recruiting chromatin-remodeling factors such as histone deacetylases and histone methyltransferases. pRB binds primarily to E2F1-3, inhibiting their activity in G1. The other two pocket proteins, p107 and p130, are also involved in regulation of the E2Fs. For example, E2F4 associates with the three different pocket proteins in different parts of the cell cycle. E2F5 is primarily regulated by p130. (9)
Protein Degradation
The ubiquitin/26S proteasome proteolytic pathway is highly conserved in all eukaryotes and is involved in many important cellular functions, including the control of the cell cycle (for review see 20). Conjugation of ubiquitin to a protein targeted for degradation involves a cascade of three enzymes: E1, E2, and E3. The E1 ubiquitin-activating enzyme transfers activated
ubiquitin to a ubiquitin-conjugating enzyme (E2). The transfer of ubiquitin to the target protein substrate usually requires an ubiquitin-ligase activity (E3).
Figure 5: Schematic representation of ubiquitin/26S proteasome proteolytic pathway. The ubiquitin-activating enzyme (E1) transfers activated ubiquitin to an ubiquitin-conjugating enzyme (E2). With the help of one of many ubiquitin ligases (E3s), an E2 transfers ubiquitin to the substrate to target it for degradation by the 26S proteasome.
Two related E3s play central roles in regulating cell cycle events: the SCF (Skp1/Cul1/F-box protein) complex and the APC. SCF activity is constant during the cell cycle and the ubiquitylation of a target protein is controlled by phosphorylation of the target protein. The APC activity changes during the cell cycle and the ubiquitylation of a target protein is controlled by the APC activators Cdc20 and Cdh1. In addition, phosphorylation of the APC itself is needed for optimal APC activity.
The anaphase-promoting complex is composed of at least 11 subunits. In early mitosis, APC is activated by binding of Cdc20, in late mitosis Cdc20 is replaced by Cdh1, which keeps APC active until the end of the
subsequent G1 phase (Figure 6). Cdc20 is targeted for degradation in late ATP
+
AMP
26S proteasome
Ub Ub Ub Ub
E1 E2 E3
substrate Ub
Ub
mitosis by the APCCdh1 complex. Cdh1 binding to APC is regulated by the phosphorylation status of Cdh1. In late G1, Cdh1 is phosphorylated and can no longer bind to the APC. In addition, a Cdh1 inhibitor, Emi1, is
transcriptionally activated at the G1-S transition (21). The APC activators Cdc20 and Cdh1 recognize specific sequences in the N-terminal part of the target protein, a D-box (destruction box with the consensus sequence RxxLxxxxN) and/or a KEN box (with amino acid consensus sequence KEN). For reviews on the APC see (22) and (23).
Figure 6. The activity of Cdc20-activated versus Cdh1-activated APC during the cell cycle.
G1 S
G2
prophas prometaphas metaphas anaphas telophas
APCCdc20 APCCdh1
Activity
Ribonucleotide Reductase
In mammalian cells, the substrates of RNR are ribonucleoside diphosphates, which are reduced to the corresponding deoxyribonucleoside diphosphates.
Figure 7. Schematic representation of the reduction of a ribonucleoside diphosphate to the corresponding deoxyribonucleoside diphosphate. The reaction is catalyzed by ribonucleotide reductase.
The reduction of ribonucleotides to deoxyribonucleotides requires radical chemistry. Ribonucleotide reductases are divided into three different classes based on the mechanism they use for radical generation and on their
structure (24). The mammalian enzyme discussed in this thesis belongs to the class Ia enzymes and forms a stable tyrosyl radical. The R2 RNR subunit contains a binuclear iron center, which generates the stable tyrosyl radical necessary for catalysis. The radical is transferred to the catalytic site, located in the R1 subunit, during each round of catalysis (25). The R1 subunit also contains two allosteric sites: the specificity site and the activity site. The specificity site regulates the balance among the four dNTP pools by binding either ATP, dTTP, dGTP, or dATP. The activity site regulates the total dNTP pool size by monitoring the dATP/ATP ratio (26). When the dATP pool reaches a certain level, RNR activity is shut off by dATP
feedback inhibition.
NDP dNDP
base base
Figure 8. Schematic representation of mammalian ribonucleotide reductase (courtesy of Andrei Chabes).
Transcription of both R1 and R2 genes is activated with the onset of S phase. There is no detectable promoter activity, R1/R2 mRNA or enzyme activity in resting cells. After cells have entered S phase, both R1/R2 mRNAs and enzyme activity can be detected (2). Although the R1 and R2 genes are transcriptionally activated at the same point during the cell cycle, their promoter structures appear completely different. Sequence similarities between mouse and human R1 promoters provided a useful tool for
elucidation of the S phase-specific transcriptional regulation of the R1 gene.
The R1 promoter contains an initiator element, located at the transcription start site. Similarly to the TATA box, the initiator element directs the general transcription machinery to the correct initiation site on the promoter (27). The R1 initiator element, together with a downstream element, is necessary for cell cycle-regulated R1 promoter activity. Furthermore, two nearly identical upstream 23 nt elements, binding the transcription factor YY-1, are important for R1 promoter strength (28, 29). In contrast to the R1 promoter comparison, a comparison between the human and the mouse R2 promoters demonstrated few obvious homology regions except for a non- canonical TATA box with the sequence TTTAAA, and a CCAAT box in the
Large subunit (R1)
Allosteric specificity site
Catalytic site Long-range
radical transfer
Small subunit (R2)
Allosteric activity site
Tyrosyl radical
mouse promoter and three CCAAT boxes in the human promoter. The transcription factor NF-Y binds specifically to the mouse R2 CCAAT box in vitro (30). The results discussed in Part II provide further insight into the transcriptional regulation of the R2 gene.
The R1 protein has a long half-life (>20 h) and the protein levels are almost constant, and in excess, in proliferating cells (31). On the contrary, the R2 subunit is specifically degraded in mitosis. The degradation of the R2 protein regulates RNR activity both during the normal cell cycle and in response to DNA damage and replication blocks (3). The R2 protein is uniquely phosphorylated on Ser20 by cell cycle-dependent kinases both in vivo and in vitro (32, 33). This phosphorylation does not, however, affect the stability of the R2 protein (3). Treatment with the proteasome inhibitor LLnL results in stabilization of the R2 protein. This result suggests the involvement of the ubiquitin/26S proteasome-dependent pathway. In
addition, a putative destruction box was identified in the R2 protein between amino acids 5 and 13. However, a protein lacking the first 20 amino acids has the same stability as the wild type protein (3). A mechanism for the degradation of the R2 protein is described in Part II.
There are many findings underscoring the importance of ribonucleotide reductase regulation. The strict regulation of RNR on multiple levels keeps the dNTP pools in balance – a necessity for the normal replication and repair of DNA. A failure in the control of the dNTP levels and/or their relative amounts leads to cell death or genetic abnormalities (1). Also, deregulated expression of R2, and accordingly deregulated RNR activity, can increase the malignancy of tumors (34). Ribonucleotide reductase inhibitors are used successfully in clinics as antiproliferative treatment.
Such drugs include, hydroxyurea (HU), a radical scavenger targeting the tyrosyl radical in the R2 protein (35). Another example of a therapeutic agent is peptidomimetic, which interferes with the interaction between the herpes simplex virus R1 and R2 subunits (36). Thus the study of RNR regulation will hopefully reveal new strategies for control of human malignancies. In a broader sense, studying ribonucleotide reductase, a key enzyme in DNA metabolism, and understanding its regulation, both on the level of transcription and protein stability, can increase our knowledge about the mechanisms that control DNA synthesis, DNA repair and cell division.
Part II. Results
In this part I describe what we have learnt about the transcriptional
regulation of the R2 gene and the M phase-specific R2 protein degradation based on the results presented in this thesis.
1. Transcriptional regulation of the R2 gene.
The mouse R2 promoter contains a CCAAT box and a TATA box with the non-canonical sequence TTTAAA (37). We first focused on these two elements. The CCAAT box binds transcription factor NF-Y and has been implicated in the transcriptional regulation of the R2 gene (30).
Paper I: Using a luciferase reporter gene construct with a mutated CCAAT box, we verified the importance of the CCAAT box for the strength of the mouse R2 promoter. The S phase specificity, however, was unaffected by the CCAAT box mutation. This result was different from previously reported results where the S phase specificity of the R2 promoter also was affected by mutation of the CCAAT box (30). The difference is most likely due to unspecific background activity of the luciferase reporter gene
backbone vector used in the previous study. In collaboration with the laboratory of Stefan Björklund, the CCAAT box mutation was also investigated in different in vitro systems. Corresponding results were obtained in a mouse cell nuclear extract in vitro transcription system.
However, in an in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF, no difference in activity could be detected between the wild type and the CCAAT-mutated promoter. The CCAAT box redundancy in the in vitro reconstituted system can be explained by the lack of transcription factor NF-Y. However, addition of recombinant NF-Y did not restore the
CCAAT-box dependency of the R2 promoter. Additional players appear to be needed to restore transcriptional activation above basal levels. This result suggests that the reconstituted in vitro system is more suitable for studying the assembly of the basal transcription machinery, which is demonstrated in paper II.
Paper II: Next we turned our focus to the TATA box. The TATA box consensus sequence is TATA(A/T)A(A/T). It is known, however, that the TATA-box binding protein (TBP) can interact with sequences that differ considerably from this consensus sequence (38). A first step in the study of the mouse R2 promoter TATA box was to verify the TBP binding to the non-canonical sequence TTTAAA. Indeed, we could demonstrate the binding of TBP to the mouse R2 TATA box by electrophoretic mobility shift analysis (EMSA).
Sequence-specific DNA-binding small molecules that can permeate cells could potentially regulate transcription of specific genes. Polyamides composed of imidazole, pyrrole, hydroxypyrrole, and β-alanine, are synthetic ligands that can be designed to bind predetermined DNA
sequences. These minor-groove DNA-binding molecules block eukaryotic transcription factors from binding to their cognate DNA sequences and inhibit transcription, both in vitro and, in a few cases, in cell culture experiments (39, 40). In an attempt to develop a proliferation inhibitor, a polyamide binding to the adjacent region of the mouse R2 promoter TATA box was designed by the laboratory of Peter Dervan (California Institute of Technology). In theory, the polyamide could block the transcription of the R2 subunit and, as a consequence, the cells would be arrested in early S phase of the cell cycle due to lack of ribonucleotide reductase activity.
The designed polyamide was tested in vitro in EMSA and proved to block the binding of TBP to the R2 TATA box. In addition, the polyamide was added to asynchronous Balb/3T3 cells stably transfected with a reporter gene construct where the luciferase gene is under the control of the full- length mouse R2 promoter (3). There was, however, no obvious effect on the R2-promoter luciferase expression. To test if the TATA box was
redundant for transcriptional activation of the R2 gene, a luciferase reporter- gene construct with a mutated TATA box sequence was tested in a
luciferase assay. To our surprise, but in accordance with the previous experiment, the R2 promoter activity was only slightly decreased by the TATA box mutation. In collaboration with the laboratory of Stefan Björklund, the mutated TATA box R2 promoter was investigated in a reconstituted in vitro system. During these studies it became evident that there was a difference in the TATA box-dependency of the R2 promoter based on whether 17 bp of the 5’ untranslated region (5’UTR) were present in the promoter construct or not. This result was also confirmed in vivo with luciferase reporter-gene assays. A palindromic sequence within the first 17 bp of the 5’ UTR appears to interact with TFIID and in this way eliminate the need for the atypical TATA box in the R2 promoter. However, mutating the 5’ UTR had an activating effect suggesting that a TATA-box dependent R2 promoter lacking the 5’ UTR has a more efficient initiation of
transcription. The exact mechanism of the assembly of the general transcription machinery on the core R2 promoter needs to be further elucidated.
Paper III: To identify elements responsible for the S phase specificity of the R2 promoter, a promoter sequence comparison between different species
was conducted. In addition to the already cloned and sequenced mouse and human promoters, the guinea pig R2 promoter region was cloned and sequenced. Additionally, DNase I footprinting assays were performed to identify protein-binding regions in the mouse R2 promoter. Finally, since the E2F transcription factor family is known to be involved in regulating many S phase-specific genes, and had been implicated in the regulation of the R2 transcription (10), we identified putative E2F-binding elements in the R2 promoter by sequence analysis. Using these three approaches, we
identified a protein-binding region located ~ 500 bp upstream from the transcription start site, and four putative E2F binding sites.
Mutation analysis of these elements demonstrated that the upstream region is necessary for full promoter activity. The exact sequence in this region that is necessary and sufficient to provide full R2 promoter activity still needs to be elucidated. The S phase-specific pattern of the R2 gene expression is most likely due to E2F-mediated repression of the promoter activity in G1. One of the four putative E2F sites was identified as being responsible for mediating this repression. This E2F site is highly conserved in mouse, human and guinea pig R2 promoters, while the other putative E2F sites are not clearly conserved.
Chromatin immunoprecipitation experiments demonstrated that E2F4 binds to the R2 promoter in vivo. As was pointed out in the introduction, E2F4 is one of the repressive E2Fs and the most abundant in the nucleus during G1 phase, while in S phase most of the E2F4 is cytoplasmic. This suggests a model where E2F4 binds to the R2 promoter in G1 phase and blocks its transcription. At the G1/S transition, the pocket proteins are phosphorylated, E2F4 comes off the promoter and R2 gene transcription is activated.
Interestingly, one study has demonstrated that the R2 gene expression was de-repressed in G1 in primary cells lacking the two pocket-proteins p107 and p130, the primary partners of E2F4 in G1 phase (41). This study also showed that the S phase specificity of the R2 gene expression was not affected in pRB null cells. The activating E2Fs, E2F 1 to E2F3, are mainly associated with pRB, while E2F4 is associated with all different pRB family members. Taken together, these findings support the involvement of E2F4 in the regulation of the R2 promoter.
The exact mechanism of E2F-mediated repression of the R2 promoter is not known and needs to be studied further. One mechanism often associated with E2F-mediated repression is the recruitment of histone-modifying enzymes. The level of chromatin formation in transient transfections is not fully understood. There is at least one study suggesting that small amounts of transiently transfected plasmids are indeed covered with nucleosomes (42). Because our studies were conducted using transient transfection experiments, it is difficult to know if recruitment of histone-modifying enzymes is the primary mechanism or not. It would be interesting to study the E2F site mutated R2 promoter in a defined chromatin context. One way to do this would be stable transfection experiments, where the reporter construct is integrated in the genome and as a consequence organized into chromatin.
Several studies have shown that treatment with the specific HDAC inhibitor trichostatin A (TSA) only relieves repression of some E2F-repressed
promoters (43). Also, recruitment of the pRB-binding protein, RPB1, to promoters results in both HDAC-dependent and independent repression (44). This suggests HDAC-independent mechanisms for E2F-mediated
repression. There are some histone modifying enzyme-independent mechanisms reported. For example, one study suggests that E2F recruits TFIID to the promoter and that this recruitment is blocked by pRB (45).
We have preliminary results showing that cells transfected with R2 promoter luciferase reporter-gene construct and treated with TSA, display no relief of the G1 repression in a luciferase assay, supporting a HDAC- independent mechanism (data not shown).
A possible mechanism for the E2F-mediated R2 promoter repression could be E2F4 interfering with the binding of NF-Y to the CCAAT box, which is situated adjacent to the E2F site. However, mutation studies where both these sites were mutated simultaneously do not support this mechanism due to the following observations: A promoter with a mutated E2F site has an increased activity, while a promoter with a mutated CCAAT box has a severely decreased activity. Combining the two mutations results in a promoter with increased activity, even though NF-Y cannot bind to and activate the promoter. If blocking of NF-Y binding were indeed the mechanism for E2F-mediated repression, a dual mutation should have resulted in an inactive promoter similar to when the CCAAT box is mutated alone.
A study by Zhou and Yen of the human promoter has suggested that the CCAAT boxes are responsible for the S phase specificity of the human R2 promoter (46). This report contradicts our results with the mouse R2 promoter. There are several possible explanations for this discrepancy, the simplest being a species-specific promoter regulation. However, when we stably transfected the human R2 gene into mouse cells, and the mouse R2 gene into human cells, we were able to observe R2 gene expression in both
cases (paper III). These results show that the human R2 promoter is active in mouse cells and reciprocally the mouse R2 promoter is active in human cells, indicating that the promoter regulation is conserved. Therefore, the discrepancy is probably not due to species-specific regulation. There are, however, other possibilities. For example, the longest reporter construct in the Zhou and Yen study includes the first 659 bp upstream of the
transcription start site, basically only covering the three CCAAT boxes. As a consequence, the influence of an upstream region, which we showed to be important for the mouse R2 promoter activity, is not investigated. The corresponding putative regulatory upstream region in the human promoter is located approximately 1300 bp upstream of the transcription start site based on sequence comparisons between the mouse, human, and guinea pig R2 promoters. The sequence analysis showed several conserved sequences in the upstream region between all three promoters (paper III). Since the reporter constructs in the Zhou and Yen report is relatively short compared to the corresponding mouse constructs used in our studies, it is possible that they never obtained full promoter activity. It is also interesting to note that the Zhou and Yen study uses constructs where the transcription start site is fused directly to the luciferase gene. Therefore, the 5’ untranslated region present in the endogenous promoter and that is involved in the assembly of the general transcription machinery on the mouse R2 promoter (paper II) is not present. The Zhou and Yen constructs are most likely dependent on the non-canonical TATA box and are not regulated in the same way as the wild- type R2 promoter is regulated according to our findings (paper II).
In conclusion, the positioning of the basal machinery on the mouse R2 promoter is not primarily aided by binding of TBP to the TATA box.
Additional contacts by TFIID to the core promoter, including sequences
downstream from the transcription start site, are involved. The binding of NF-Y to the CCAAT box is important for the strength of the mouse R2 promoter. In G1 phase of the cell cycle, the R2 promoter activity is low due to E2F-mediated repression. An upstream region is needed for full mouse R2 promoter activity.
2. Regulated protein degradation of the R2 protein
The R2 RNR subunit is degraded in mitosis. A previous study demonstrated that treatment with the proteasome inhibitor LLnL resulted in stabilization of the R2 protein (3). This suggested the involvement of the ubiquitin proteasome dependent pathway. In 2001, Pfleger and Kirschner identified a novel recognition signal for the anaphase-promoting complex, the KEN box (47).
Paper IV: A putative KEN box is located at position 30-32 from the N- terminus of R2. It is completely conserved, both in position and amino acid residues, in the human, mouse, hamster, and guinea pig R2 proteins. Also, an N-terminally located KEN sequence can be found in the R2 protein homologues present in Caenorhabditis elegans and Drosophila, but not in budding or fission yeast. Mutation of the KEN sequence in the R2 protein resulted in a stabilized R2 protein when overexpressed in mouse cells. The R2 protein stability was monitored during the cell cycle and the KEN
mutant R2 protein was present in late mitosis and the following G1 phase. In contrast, the wild-type R2 protein overexpressed in mouse cells was
degraded in late mitosis/early G1 phase as expected.
In collaboration with Cathie Pfleger and Marc Kirschner (Harvard Medical School), the mutated KEN protein was also tested in an in vitro
ubiquitylation assay. The assay demonstrated that mutation of the KEN sequence abolished the in vitro ubiquitylation of the R2 protein. In addition, pull-down assays using in vitro-translated Myc-tagged Cdh1 demonstrated interactions between the wild-type R2 protein and Cdh1, but not between the mutated R2 protein and Cdh1. Cdc20 was also tested for interactions with the R2s, but no interaction was detected. Previous results have
suggested that the R2 degradation would take place earlier in mitosis than what would be expected for APCCdh1-dependent proteolysis. This suggestion was based on the finding that R2 is already degraded in metaphase cells, arrested by nocodazole (3). The reason for this discrepancy is not known.
We suspect that disappearance of R2 protein in nocodazole treated cells could be attributed to the loss of cells during treatment. We will further investigate this discrepancy.
In conclusion, we have identified the signal responsible for the M phase- specific degradation of the R2 protein, which in turn regulates the presence of the ribonucleotide reductase enzyme during the cell cycle. The R2 degradation impedes deoxyribonucleotide production during G1 and accordingly could be one of the mechanisms to protect the cell against unscheduled DNA synthesis.
Acknowledgements
I would like to thank everybody at the Department of Medical Biochemistry and Biophysics for all help and for making it such a nice and friendly place to work.
In addition, I would especially like to thank:
My supervisor Lars Thelander: for all your help and encouragement from the very
beginning of my PhD and all the way to the end. I especially appreciate the way you helped me to make the decision to become a PhD student, without the understanding and support I received from you I would not have dared to try. I also greatly appreciate that you gave me the opportunity to accompany Andrei to Cold Spring Harbor Laboratory!
Winship Herr (Cold Spring Harbor Laboratory): for welcoming me into your laboratory, and for helpful advice and support during the writing of this thesis.
Former and present friends in the Thelander group:
Margareta Thelander: for all your help and encouragement. I really enjoy all our interesting conversations!
Pelle Håkansson: for being a friend in the lab with whom you can share the pros and cons of being a PhD student…
Kerstin Hjortsberg: for all your help, sharing of reagents and DNA sequencing.
Anders Hofer: for all your help, especially in the beginning.
Vladimir Domkin, Olivier Guittet, Arthur Fijolek and Ulrika Rova: for help and for adding to the nice atmosphere of the lab.
Ingrid Råberg: for always helping with all the paperwork in your competent and nice way.
Lola Fredriksson: for making all the solutions and keeping the lab functional.
Stefan Björklund and Irina Kotova: for great collaborations and helpful discussions.
Sergei Lobov, Magnus Hallberg, Sven Carlsson, and Richard Lundmark: for help and lending of equipment.
Urban Backman: for taking care of all problems during teaching.
My friends in the Herr lab: for all your encouragement and help.
My family and friends: for support and lots of fun!
My wonderful parents: your support, encouragement, persuasion, and belief in my ability have made this thesis possible!
Andrei: for sharing your wisdom, knowledge, love and life with me.
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