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1.
Figure 7.

Figure 7. From: Regulation of neuron survival and death by p130 and associated chromatin modifiers.

A model for the mechanism by which neuron survival and death are regulated by p130 and associated chromatin modifiers.

David X. Liu, et al. Genes Dev. 2005 March 15;19(6):719-732.
2.
Figure 4.

Figure 4. From: Regulation of neuron survival and death by p130 and associated chromatin modifiers.

p130 associates with HDAC1 and Suv39H1, and this association is lost in response to apoptotic stimuli. (A) Cellular association between endogenous p130 and Suv39H1. IPs were prepared from whole-cell extracts of neuronal PC12 cells using the indicated antibodies. Irr is an irrelevant (control) antibody (p53). IPs were subjected to Western blotting with antibodies against p130 and Suv39H1 as indicated. (B,C) Apoptotic stimuli lead to loss of cellular association between endogenous p130 and HDAC1 (B) or Suv39H1 (C). IPs were prepared from extracts of cortical neurons treated ±Cpt for 9 h using anti-HDAC1 (B) or of neuronal PC12 cells undergoing NGF withdrawal for 0, 8, and 18 h using anti-Suv39H1 (C). The IPs were subjected to Western immunoblotting using anti-p130 and anti-HDAC1 (B) or anti-Suv39H1 (C). (D,E) Cpt treatment leads to loss of association between p130 and H3-HMTase activity. IPs were prepared as in B using antibodies against either p53 (Irr, irrelevant) or p130. Half of each IP was used for fluorographic analysis (D) and the other half for H3-HMTase assay (E). In D, reaction products were separated by PAGE and subjected to fluorography. Loading of H3 histone is shown by Ponceau Red staining. In E, reaction products were spotted on filters and processed as described in Materials and Methods. (F) As in E except that IPs were prepared from extracts of neuronal PC12 cells undergoing NGF withdrawal for 0 and 16 h.

David X. Liu, et al. Genes Dev. 2005 March 15;19(6):719-732.
3.
Figure 6.

Figure 6. From: Regulation of neuron survival and death by p130 and associated chromatin modifiers.

Neuronal survival is regulated by chromatin modifiers tethered to DNA by E2F4-p130. (A) Association of HDAC1 and Suv39H1 with the B-myb promoter is regulated by an apoptotic stimulus and requires anchored interaction with E2F4-(hypophosphorylated) p130. Neuronal PC12 cells were transfected with the indicated Flag-tagged constructs and 24 h later treated for 4 h ±Cpt as indicated. Twenty micrograms of cross-linked chromatin per sample was subjected to ChIP with 4 μg of anti-Flag (M2) antibody. The IPs were each divided into two equal aliquots. One (1st IP: FLAG; upper panel) was de-cross-linked, and 1/10 of this was analyzed by 4%-12% gradient SDS-PAGE and immunoblotted with the indicated antisera. Half of each of the same aliquots was used to extract DNA that was analyzed by PCR for detection of B-myb promoter. The second aliquot was subjected to a second IP with 2 μg of anti-HDAC1 (2nd IP, HDAC1; lower panel). After de-cross-linking, 1/10 of the IP was analyzed as above for HDAC1, 50% was used for PCR detection of B-myb promoter as above, and 40% to measure HMT activity as in Materials and Methods. (B) Derepression of the B-myb promoter by mutant E2F4-p130 fusion proteins compromised for interaction with corepressors. Neuronal PC12 cells were transfected with indicated constructs and processed as in . (C) Induction of neuron death by mutant E2F4-p130 fusion proteins compromised for interaction with corepressors. Cortical neurons were cotransfected with the indicated constructs, and eGFP and the number of transfected (eGFP+) cells were counted as in . (D) Phosphorylation-resistant E2F4-p130 fusion proteins protect neurons from apoptotic death. Cortical neurons were cotransfected with the indicated constructs and eGFP. One day later, the number of eGFP-positive cells was assessed (Day 0) and Cpt was added. eGFP-positive cells were counted on each of the following 3 d as in .

David X. Liu, et al. Genes Dev. 2005 March 15;19(6):719-732.
4.
Figure 3.

Figure 3. From: Regulation of neuron survival and death by p130 and associated chromatin modifiers.

p130 and E2F4 are required for neuronal survival. (A) siRNAs against individual Rb family members down-regulate their specific targets in cortical neurons, and only p130 siRNA induces apoptosis. Cortical neurons were cotransfected with eGFP and siRNA constructs against Rb (panels a-c), p107 (panels d-f), or p130 (panels g-i). (Panels b,e,h) Two days later, cultures were fixed and immunostained as indicated for expression of Rb family members. (Panels c,f,i) Nuclei were stained with Hoechst 33342. (Panels a,d,g) Arrows show transfected cells (green). Bar, 20 μm. (B) Efficiency of indicated siRNA constructs in down-regulating Rb family members in cortical neurons. Transfection was as in A. The proportions of transfected (eGFP+) cells (35-136/culture in triplicate) expressing each antigen were assessed. (C,D) Down-regulation of p130 or E2F4, but not of Rb or p107, induces apoptosis of cortical neurons. In C, cells were cotransfected with indicated siRNA and human (Hs) constructs and eGFP and 4 h later, the transfection medium was replaced with culture medium ±BAF or Flavo as indicated. The number of eGFP+ cells was counted 24 h after transfection (day 0) and on each of the next 2 d and percent surviving cells expressed relative to that at day 0 (set arbitrarily to 100%). In D, transfection was as in C, and 2 d later cells were stained with Hoechst dye. Transfected (eGFP+) cells were scored for the presence of apoptotic nuclei. (E) Loss of p130 or displacement with mutant p130(C894F) compromises survival of cortical neurons. Cells were cotransfected with eGFP and the indicated constructs. The number of GFP+ cells in the cultures was counted as in C. (F) Loss of p130, E2F4, or expression of p130(C894F) causes derepression of a B-myb-luc reporter. Neuronal PC12 cells were cotransfected with pcDNA-lacZ, B-myb-luc, and indicated constructs. Cells were harvested 48 h later and subjected to luciferase and β-gal assays. p130(h) is a human p130 construct. (G) p130 and p130 mutated at phosphorylation sites protect neuronal PC12 cells from NGF withdrawal (W/D). Experiments were carried out and data were expressed as in E except cells were washed and anti-NGF was added 24 h after transfection.

David X. Liu, et al. Genes Dev. 2005 March 15;19(6):719-732.
5.
Figure 2.

Figure 2. From: Regulation of neuron survival and death by p130 and associated chromatin modifiers.

Apoptotic stimuli evoke loss of hypophosphorylated p130 and exodus of E2F4 from the nuclei of neuronal cells. (A) Western immunoblot analysis of p130 in whole-cell extracts of cultured cortical neurons treated ±Cpt. Blots were probed with p130 antibody C-20. Equal loadings were confirmed by ERK staining (data not shown) and the presence of nonspecific bands on the top and bottom of the blot. (B) Western immunoblot analysis of p130 isoforms in whole-cell extracts of neuronal PC12 cells at 0, 3, and 18 h after NGF withdrawal. (C) Conversion of hyperphosphorylated p130 (pp130) into hypophosphorylated p130 (p130) by λ-phosphatase (Ptase). (D) Western immunoblot analysis of p130 isoforms (p130, hypophosphorylated; pp130, hyperphosphorylated) in nuclear (N) and cytosolic (C) fractions of neuronal PC12 cells. Actin and PCNA are cytosolic and nuclear markers, respectively. (E,F) Western immunoblot analysis of p130 levels in nuclear (D) and cytosolic (E) fractions of neuronal PC12 cells treated with Cpt for the indicated times. The presence of Flavo is indicated by +F. (G) E2F4 bound to the endogenous B-myb promoter specifically associates with p130, but not Rb or p107 in neuronal PC12 cells. Neuronal PC12 cells were subjected to ChIP using 30 μg of cross-linked chromatin and 30 μg of Agarose-conjugated anti-E2F4 antibody as described in Materials and Methods. Bound material was eluted and equal aliquots were subjected to IP with antibodies against either Rb, p107, or p130. (Top panel) One-tenth of each pellet was de-cross-linked in sample buffer, and the samples were resolved by SDS-PAGE on a 4%-12% gel and analyzed by Western immunoblotting with anti-p130. (Bottom panel) Half of each pellet was used to extract DNA that was subjected to PCR to detect B-myb promoter sequence. The supernatants of the first IPs were subjected to a second round (2nd IP) with the same antibodies to ensure that the first round had reached completion. As positive controls, a fraction (1.7%) of the eluate from the ChIP with anti-E2F4 was loaded into the far-left lane (E2F4), while the same fraction from the chromatin before IP was loaded into the far-right lane (input). (H) Change in distribution of E2F4 from nuclear (N) to the cytosolic (C) fraction in cortical neurons after Cpt treatment.

David X. Liu, et al. Genes Dev. 2005 March 15;19(6):719-732.
6.
Figure 1.

Figure 1. From: Regulation of neuron survival and death by p130 and associated chromatin modifiers.

p130 is the major Rb-family member complexed with E2F in neurons, and such complexes are lost in response to apoptotic stimuli. (A) EMSAs on extracts of cortical neurons and the E2F-binding sequence from the adenovirus E2 promoter () (lanes 1-4) or rat B-myb promoter sequence (lanes 5-8). Samples in lanes 1-4 were incubated without (control) or with anti-p130 (Transduction Laboratories), p107 (Santa Cruz C-18), or Rb (Oncogene Science), and in lanes 5-8 with Santa Cruz antisera C-20 (p130), C-18 (p107), and C-15 (Rb) and control antibody p27 (C-19). The solid arrow marks the E2F-p130 complex that is selectively supershifted by anti-p130. The open arrow indicates free E2F. Irrelevant lanes were removed from the original image. (B) ChIP assays on neuronal PC12 cells stably transfected with the cdc25A-luc reporter and treated for 9 h ±Cpt. Chromatin from the cells was subjected to IP with the indicated antibodies, and the relative amount of promoter in the IPs (pellet) was quantified by PCR and reported as relative promoter occupancy (the value for the p130 pellet at 0 h was arbitrarily set at 100%). Supernatants from the IPs were subjected to a second round of IP/PCR to verify that the first round of IP was complete. “Mock” indicates values for ChIP assays on samples in which antibodies were omitted. The data here and all other quantified experiments below are means of values (±SEM) from three to six independent experiments. (C) ChIP assays carried out as in B except that cells were stably transfected with the B-myb-luc reporter. Times of exposure to Cpt and Flavo are as indicated. Data were quantified and expressed as in B. (D) ChIP assays were carried out as in C using p130 antibody and chromatin from neuronal PC12 cells transfected for 2 d with the indicated promoters and exposed as indicated to Cpt and Flavo. The gel shows PCR on recovered DNA using primers for mouse B-myb (lanes 1-6) and human cdc25A (lanes 7-10) promoters. (Lanes 5,6) PCR results for controls containing 5% of the pre-IP input for wild-type and mutant mB-myb-luc. (Lane 10) Ten percent of the pre-IP input for cdc25A-luc (hscdc25A). (E) As in D except that chromatin was prepared from cortical neurons treated ±Cpt as indicated. IPs were carried out with a control antibody (p27) in lane 1 and with anti-p130 in lanes 2-5. For lane 6, the input was 10% of the pre-IP DNA. PCR was carried out using either primers for the rat B-myb (upper panel) or actin (lower panel) promoters. (F) EMSA on extracts of cortical neurons (lanes 1-4) or neuronal PC12 cells (lanes 5-9) treated with Cpt for the indicated times in the absence or presence of Flavo (+F). The commercial E2F consensus DNA sequence was used. (G) As in F except that extracts of SCG neurons undergoing NGF withdrawal (W/D) for the indicated times ±Flavo (F) were used.

David X. Liu, et al. Genes Dev. 2005 March 15;19(6):719-732.
7.
Figure 5.

Figure 5. From: Regulation of neuron survival and death by p130 and associated chromatin modifiers.

Relationship between chromatin modification and neuron survival and death. (A) Expression of truncated Suv39H1 or si-Suv39H1 leads to gene derepression of an E2F-regulated construct and evokes death. Neuronal PC12 cells were cotransfected with pcDNA-lacZ, B-myb-luc (right), or with eGFP (left) and the indicated Suv39H1 constructs. (Left) Cells were harvested 2 d later and assessed for relative luciferase activity as in . (Right) The number of eGFP-positive cells was assessed as in . Significance comparison to the vector on same day: () p < 0.03. (B) Inhibition of HDAC activity leads to derepression of an E2F-regulated construct and evokes neuron cell death. (Left) Neuronal PC12 cells were cotransfected with pcDNA-lacZ and B-myb-luc constructs. Two days later, cells were exposed to 20 nM TSA for 24 h. Luciferase activity was measured as in . (Right) Cortical neurons were treated with or without 20 nM TSA, and survival was assessed. Cell counts before the addition of TSA on 3 DIV (Day 0) were set to 100%, and survival was assessed as in . Significance comparisons on same day: () p < 0.01. (★★) p < 0.001. (C) An apoptotic stimulus leads to selective modification of chromatin associated with E2F-binding sites. ChIP assays were carried out using material from PC12 cells stably transfected with wild-type or mutant cdc25A-luc (mutant for E2F binding) (left) or wild-type PC12 cells (right). Cells were treated for indicated times with Cpt, and chromatin samples were subjected to IP with saturating amounts (6 μg/assay) of anti-Ac-p-H3 antibody. Real-time PCR (including Mock setup) was carried out as in . Values are normalized so that the level of promoter detected for the Mut cdc25A or Actin after 9 h of Cpt treatment equals 100. (D) p130-associated corepressors regulate histone modification at the endogenous B-myb promoter. ChIP assays were carried out as in except that neuronal PC12 cells were transfected with the indicated constructs or treated ±20 nM TSA. Cross-linked chromatin was prepared as in Materials and Methods 24 h after transfection or TSA treatment. Forty micrograms per transfected sample was subjected to ChIP with 8 μg of agarose-conjugated anti-HA (left) or anti-Myc (middle). The IPs were eluted by three rounds of treatment with the Pro-Found Co-IP Kit (Pierce) according to the manufacturer's instructions and each was divided into four equal aliquots, subjected to a second ChIP using antibodies as indicated. (Right) In the case of cells ±TSA, a single ChIP was carried out using anti-MeH3 or anti-Ac-p-H3. DNA was recovered from the IPs as in Materials and Methods, and an equal amount (25%) was used for PCR detection using primers specific to the rat B-myb promoter. As positive controls for PCR, 0.5 μg of chromatin from each sample before HA-Flag-IP was included in lanes 1 and 5, respectively.

David X. Liu, et al. Genes Dev. 2005 March 15;19(6):719-732.

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