Idea based on initial experiment from (http://www.nature.com/ncb/journal/v9/n2/abs/ncb1532.html) which spawned entire study.
Question: What co-factors are also bound to ppRB-E2F1 complexes? Are different subunits bound under different cellular contexts?
1) Purify ppRBΔG-E2F1 complexes (from pRBΔG MEFs or other cell types) from nuclear extract prepared during different cellular contexts where ppRB is enriched:
i) G1 arrest
ii) DNA Damage
iii) Post-mitotic (senescent) state
Steps for ppRBΔG-E2F1 complex purification:
i) GST-pRB affinity column
ii) IP: anti-E2F1
2) Mass spectrometry used to identify previously undiscovered subunits bound to ppRBΔG-E2F1 complex. Identity of subunits will provide leads for further investigation of physiological purpose of ppRB-E2F1 complexes formed through the pRB C-terminal E2F1-specific site.
Question: What co-factors are also bound to ppRB-E2F1 complexes? Are different subunits bound under different cellular contexts?
1) Purify ppRBΔG-E2F1 complexes (from pRBΔG MEFs or other cell types) from nuclear extract prepared during different cellular contexts where ppRB is enriched:
i) G1 arrest
ii) DNA Damage
iii) Post-mitotic (senescent) state
Steps for ppRBΔG-E2F1 complex purification:
i) GST-pRB affinity column
ii) IP: anti-E2F1
2) Mass spectrometry used to identify previously undiscovered subunits bound to ppRBΔG-E2F1 complex. Identity of subunits will provide leads for further investigation of physiological purpose of ppRB-E2F1 complexes formed through the pRB C-terminal E2F1-specific site.
A Different Complex for Every Context: The Role of Specific Site in RB-mediated G1 arrest:
Question: What role does the specific site play in pRB mediated cell cycle arrest? If any, is this dependent on the interaction with E2F1, or another binding partner (i.e. Skp2)? Previos SaOS-2 cell cycle arrest assays conducted with various transfected pRB mutants demonstrate that disruption of E2F binding via pRBΔG results in only a partial defect in pRB mediated cell cycle arrest. When abrogation of E2F binding is combined with other mutations (pRBΔG+ΔCRF or pRBΔG+ΔS), an increased disruption of pRB mediated cell cycle arrest is observed, indicating that interactions through the other mutated contact points also play a role in pRB mediated cell cycle arrest. pRBΔCRF disrupts binding of chromatin remodeling factors, as well as Cdh1, which is required to form the pRB-APC-Cdh1-Skp2 triple complex that stabilizes p27Kip1 to allow for E2F-independent pRB mediated cell cycle arrest. pRBΔS is known to disrupt binding to E2F1, but may also disrupt binding of Skp2. If pRB cannot bind E2Fs or Skp2, then both E2F-dependent and independent mechanisms cannot be utilized by pRB.
I suspect that the intact general E2F binding site of pRBΔS will allow for pRB-mediated cell cycle arrest with efficacy parallel to that of wild-type pRB. Is it possible that even with an intact general E2F binding site, under certain cellular contexts pRB requires the pRB-APC-Cdh1-Skp2 triple complex for pRB mediated G1 arrest? If so, pRBΔS cells should be defective for proliferative control during such cellular contexts which have yet to be defined. Perhaps these cellular contexts arise during development. If so, what developmental defects might be expected in Rb1ΔS /ΔS mice? The expectations may be convoluted due to the dual role that this C-terminal region may mediate. Failure to attenuate proliferation combined with hyper-sensitivity to E2F1-dependent apoptosis cannot produce any defects since the two would cancel each other out. Of course, this cannot be the case as pRB cannot execute two mutually exclusive effects simultaneously. Even if E2F1 and Skp2 bindng to the pRB C-terminus are not mutually exclusive in vitro, they must be within physiological contexts due to the opposing roles the play in proliferation vs survival.
I believe under certain physiological contexts, different post translational modifications are imposed on pRB, E2F1, and Skp2 in order to mead out which pRB complex will form according to necessity of function by the cell. For example, during DNA Damage, the cell acts to prevent DNA replication until repair mechanisms can be executed. After attempts at repair are exhausted, an altruistic apoptotic response is executed to prevent the propogation of genomic insults through clonal expansion.
Thus, in the context of DNA damage, hypophosphorylated pRB first acts to induce G1 arrest. Is this arrest E2F-dependent or independent? If it is E2F dependent, pRB-E2F complexes should be enriched. If it is E2F independent, pRB-APC-Cdh1-Skp2 complexes should be enriched, and high levels of p27Kip1 should be detected. Is it possible that method of G1 arrest in response to DNA damage is cell type specific, or dependent on stage of development?
Second, after exhausting attempts to repair the genomic insult, the cell must be sensittive to a pro-apoptotic response. pRB-E2F1 complexes have been shown to be transcriptionally repressive regardless of which site pRB uses to bind E2F1. Thus, DNA damage should result in a decrease in pRB-E2F1 complexes in parallel with an increase in free transcriptionally active E2F1 which can induce expression of transcriptional activators of components of the apoptotic circuitry (i.e. p73). E2F1 also directly activates key components of the apoptotic caspase cascade (ex. Apaf-1, Caspase-7, FOLLOW UP), as well as components of both the intrinsic and extrinsic apoptotic pathways (bcl-2 family members) which both converge to induce the apoptotic caspase cascade.
My project aims to uncover the story of how pRB uses the specific site to control the life and death decisions of the cell. My immediate task will be to design experiments to answer the sequential list of questions which comprise the larger story. Experimental design will require that I spend time developing the tools required to execute these experiments. Developing and maintaining an in vivo mouse model, developing a system for co-expression and purification of E2F-DP1 heterodimers.
Analysis of E2F1 itself is also essential to this story. To identify which post-translational modifications allow or prevent association with the specific site, I will generate DP1-E2F1 dimers, comprised of various E2F1 mutants with alterations to specific residues in order to prevent or mimic the presence of a post-translational modification. This will lend further to define under which cellular contexts E2F1 uses the specific site to form pRB-E2F1 complexes. I will express and purify HA-tagged DP1-E2F1 dimers, and use them in pulldown experiments to identify which E2F1 mutants can or cannot bind pRB. Again, this will provide sight into which cellular contexts promote formation of pRB-E2F1 complexes through the specific site.
The emphasis is truly on cellular context: Different complexes for different cellular contexts. Are these complexes always transcriptionally repressive? Numerous studies suggest transcriptionally active pRB-E2F1 complexes can be found at pro-apoptotic promoters
While entertaining the theme of cellular context, the context of tumorigenesis cannot be overlooked. In the majority of human cancers, pRB is inactivated through upstream targeting (ex. p16Ink4a) in a manner which renders pRB constitutively hyperphosphorylated to allow for uncontested E2F-driven proliferation. In a hyperphosphorylated state, pRB can remain bound to E2F1. I believe E2F1 bound through the general site possesses different post-translational modifications from E2F1 bound to the specific site, and these modifications direct which region is utilized on pRB in a mutually exclusive mannor. I believe these post-translational modifications determine whether E2F1 is bound to promoters of cell-cycle targets, or apoptotic targets. I believe that E2F1 modified to bind to promoters of cell-cycle targets is compatible to bind to the A-B interface of pRB, and remains unbound by ppRB due to the conformational changes imposed by CDK-mediated phosphorylation of pRB. This form of E2F1 is incompatible with binding to the pRB C-terminus. Conversely, E2F1 modified to bind to promoters of apoptotic targets is compatible to bind the E2F1-specific site of the pRB C-terminus, even after CDK phosphorylation of pRB. In the context of pRB inactivation through constitutive CDK phosphorylation, "pro-proliferation" E2F1 remains unbound and transcriptionally active, while "pro-apoptotic" E2F1 remains bound and repressed. If it is the case that some ppRB-E2F1 complexes that form through the specific site can be transcriptionally active, it is necessary to define other binding partners which comprise the subunits of the entire complex to define a transcriptionally active vs repressive ppRB-E2F1 complex formed through the specific site. In the context of tumorigenesis, the cell must select for the transcriptionally repressive ppRB-E2F1 complex. The identity of this complex will provide insight into the mechanism of this oncogenic selection, and provide a viable target for small molecules that must prevent this complex from forming in order to free the "pro-apoptotic form" of E2F1 to kill the cell and halt clonal expansion.
S-phase 2000 Knudsen paper: (SEE Fig 8)
· VERY INTERESTING – Prolonged CDDP treatment - Unlike Rb+/+ MEFs which arrest but maintain % live cells without replicating (as determined by BrdU), Rb-/- MEFs do not arrest in response to CDDP, but show decrease in % live cells (as determined by FACS). It is known that checkpoint defects render tumor cells hypersensitive to antineoplastic drugs. Thus, it is possible to be unresponsive to DNA damage checkpoints, replicate damaged DNA, but still demonstrate increased sensitivity to apoptosis. Could this be the case with pRBΔS cells which may replicate damaged DNA, but demonstrate increased sensitivity to apoptosis due to an attenuated E2F1 beyond the G1 restriction point??
Question: What role does the specific site play in pRB mediated cell cycle arrest? If any, is this dependent on the interaction with E2F1, or another binding partner (i.e. Skp2)? Previos SaOS-2 cell cycle arrest assays conducted with various transfected pRB mutants demonstrate that disruption of E2F binding via pRBΔG results in only a partial defect in pRB mediated cell cycle arrest. When abrogation of E2F binding is combined with other mutations (pRBΔG+ΔCRF or pRBΔG+ΔS), an increased disruption of pRB mediated cell cycle arrest is observed, indicating that interactions through the other mutated contact points also play a role in pRB mediated cell cycle arrest. pRBΔCRF disrupts binding of chromatin remodeling factors, as well as Cdh1, which is required to form the pRB-APC-Cdh1-Skp2 triple complex that stabilizes p27Kip1 to allow for E2F-independent pRB mediated cell cycle arrest. pRBΔS is known to disrupt binding to E2F1, but may also disrupt binding of Skp2. If pRB cannot bind E2Fs or Skp2, then both E2F-dependent and independent mechanisms cannot be utilized by pRB.
I suspect that the intact general E2F binding site of pRBΔS will allow for pRB-mediated cell cycle arrest with efficacy parallel to that of wild-type pRB. Is it possible that even with an intact general E2F binding site, under certain cellular contexts pRB requires the pRB-APC-Cdh1-Skp2 triple complex for pRB mediated G1 arrest? If so, pRBΔS cells should be defective for proliferative control during such cellular contexts which have yet to be defined. Perhaps these cellular contexts arise during development. If so, what developmental defects might be expected in Rb1ΔS /ΔS mice? The expectations may be convoluted due to the dual role that this C-terminal region may mediate. Failure to attenuate proliferation combined with hyper-sensitivity to E2F1-dependent apoptosis cannot produce any defects since the two would cancel each other out. Of course, this cannot be the case as pRB cannot execute two mutually exclusive effects simultaneously. Even if E2F1 and Skp2 bindng to the pRB C-terminus are not mutually exclusive in vitro, they must be within physiological contexts due to the opposing roles the play in proliferation vs survival.
I believe under certain physiological contexts, different post translational modifications are imposed on pRB, E2F1, and Skp2 in order to mead out which pRB complex will form according to necessity of function by the cell. For example, during DNA Damage, the cell acts to prevent DNA replication until repair mechanisms can be executed. After attempts at repair are exhausted, an altruistic apoptotic response is executed to prevent the propogation of genomic insults through clonal expansion.
Thus, in the context of DNA damage, hypophosphorylated pRB first acts to induce G1 arrest. Is this arrest E2F-dependent or independent? If it is E2F dependent, pRB-E2F complexes should be enriched. If it is E2F independent, pRB-APC-Cdh1-Skp2 complexes should be enriched, and high levels of p27Kip1 should be detected. Is it possible that method of G1 arrest in response to DNA damage is cell type specific, or dependent on stage of development?
Second, after exhausting attempts to repair the genomic insult, the cell must be sensittive to a pro-apoptotic response. pRB-E2F1 complexes have been shown to be transcriptionally repressive regardless of which site pRB uses to bind E2F1. Thus, DNA damage should result in a decrease in pRB-E2F1 complexes in parallel with an increase in free transcriptionally active E2F1 which can induce expression of transcriptional activators of components of the apoptotic circuitry (i.e. p73). E2F1 also directly activates key components of the apoptotic caspase cascade (ex. Apaf-1, Caspase-7, FOLLOW UP), as well as components of both the intrinsic and extrinsic apoptotic pathways (bcl-2 family members) which both converge to induce the apoptotic caspase cascade.
My project aims to uncover the story of how pRB uses the specific site to control the life and death decisions of the cell. My immediate task will be to design experiments to answer the sequential list of questions which comprise the larger story. Experimental design will require that I spend time developing the tools required to execute these experiments. Developing and maintaining an in vivo mouse model, developing a system for co-expression and purification of E2F-DP1 heterodimers.
Analysis of E2F1 itself is also essential to this story. To identify which post-translational modifications allow or prevent association with the specific site, I will generate DP1-E2F1 dimers, comprised of various E2F1 mutants with alterations to specific residues in order to prevent or mimic the presence of a post-translational modification. This will lend further to define under which cellular contexts E2F1 uses the specific site to form pRB-E2F1 complexes. I will express and purify HA-tagged DP1-E2F1 dimers, and use them in pulldown experiments to identify which E2F1 mutants can or cannot bind pRB. Again, this will provide sight into which cellular contexts promote formation of pRB-E2F1 complexes through the specific site.
The emphasis is truly on cellular context: Different complexes for different cellular contexts. Are these complexes always transcriptionally repressive? Numerous studies suggest transcriptionally active pRB-E2F1 complexes can be found at pro-apoptotic promoters
While entertaining the theme of cellular context, the context of tumorigenesis cannot be overlooked. In the majority of human cancers, pRB is inactivated through upstream targeting (ex. p16Ink4a) in a manner which renders pRB constitutively hyperphosphorylated to allow for uncontested E2F-driven proliferation. In a hyperphosphorylated state, pRB can remain bound to E2F1. I believe E2F1 bound through the general site possesses different post-translational modifications from E2F1 bound to the specific site, and these modifications direct which region is utilized on pRB in a mutually exclusive mannor. I believe these post-translational modifications determine whether E2F1 is bound to promoters of cell-cycle targets, or apoptotic targets. I believe that E2F1 modified to bind to promoters of cell-cycle targets is compatible to bind to the A-B interface of pRB, and remains unbound by ppRB due to the conformational changes imposed by CDK-mediated phosphorylation of pRB. This form of E2F1 is incompatible with binding to the pRB C-terminus. Conversely, E2F1 modified to bind to promoters of apoptotic targets is compatible to bind the E2F1-specific site of the pRB C-terminus, even after CDK phosphorylation of pRB. In the context of pRB inactivation through constitutive CDK phosphorylation, "pro-proliferation" E2F1 remains unbound and transcriptionally active, while "pro-apoptotic" E2F1 remains bound and repressed. If it is the case that some ppRB-E2F1 complexes that form through the specific site can be transcriptionally active, it is necessary to define other binding partners which comprise the subunits of the entire complex to define a transcriptionally active vs repressive ppRB-E2F1 complex formed through the specific site. In the context of tumorigenesis, the cell must select for the transcriptionally repressive ppRB-E2F1 complex. The identity of this complex will provide insight into the mechanism of this oncogenic selection, and provide a viable target for small molecules that must prevent this complex from forming in order to free the "pro-apoptotic form" of E2F1 to kill the cell and halt clonal expansion.
S-phase 2000 Knudsen paper: (SEE Fig 8)
· VERY INTERESTING – Prolonged CDDP treatment - Unlike Rb+/+ MEFs which arrest but maintain % live cells without replicating (as determined by BrdU), Rb-/- MEFs do not arrest in response to CDDP, but show decrease in % live cells (as determined by FACS). It is known that checkpoint defects render tumor cells hypersensitive to antineoplastic drugs. Thus, it is possible to be unresponsive to DNA damage checkpoints, replicate damaged DNA, but still demonstrate increased sensitivity to apoptosis. Could this be the case with pRBΔS cells which may replicate damaged DNA, but demonstrate increased sensitivity to apoptosis due to an attenuated E2F1 beyond the G1 restriction point??