1. Volume 123, Issue 3, Pages 437-448 (November 2005)
Tumor Suppressor HIC1 Directly Regulates SIRT1 to Modulate p53-Dependent DNA- Damage Responses 
Wen Yong Chen, David H. Wang, RayWhay Chiu Yen, Jianyuan Luo, Wei Gu, Stephen B. Baylin 
Cell 
Volume 123, Issue 3, Pages (November 2005)
DOI: /j.cell
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2. Figure 1 HIC1 Mediates Apoptotic DNA-Damage Responses
(A) Hic1+/+ and Hic1−/− MEF were treated with 80 μM etoposide for 16 hr. After drug removal, survival cell numbers were measured using the XTT assay. Day 0 was the time point immediately after drug removal. The error bar is ±1SD.
(B) Examination of MEF apoptosis by TUNEL. DNA strand breaks were labeled with TMR red, and nuclei were stained with DAPI (blue). Apoptotic cells exhibited golden color as a result of color merge of these two labels.
(C and D) MCF-7 cells were infected with either Ad/lacZ or Ad/HIC1 for 24 hr and then treated with 20 μM etoposide for 16 hr. Cells were photographed at days shown, and D1 was one day after etoposide removal (C). In (D), survival of MCF-7 cells was measured by XTT assay. The error bar is ±1SD.
(E) Apoptotic DNA ladder of MCF-7 cells. Equal numbers of cells were either mock-infected or infected with Ad/lacZ or Ad/HIC1, then treated with etoposide. Two days after etoposide removal, cells (both floating and adherent) were harvested and apoptotic low-molecular-weight DNA was specifically extracted. One half of the extract from each sample was loaded per lane, and apoptotic DNA was manifested by a fragmentation ladder. Marker = nucleosome size ladder.
(F) HIC1 overexpression. Nuclear extracts of MCF-7 cells were prepared 48 hr after infection. HIC1 expression was normalized to nuclear lamin A/C, and the relative level was then compared to that of WI38 normal human fibroblasts.
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3. Figure 2 P53 Is Required for HIC1-Mediated Apoptosis
(A) MCF-7 cells were transfected with p53 shRNA or scrambled shRNA (mock) and then selected for stable integration. The p53 knockdown was monitored by Western blot. The pooled mock or p53 knockdown cells were infected with Ad/lacZ and Ad/HIC1, respectively, and then treated with 20 μM etoposide for 16 hr. Survival cell numbers were measured using the XTT assay at 2 and 4 days after the drug removal. The ratio of surviving cells with Ad/HIC1 infection to Ad/lacZ infection in mock or p53 knockdown cells was then calculated for each knockdown and plotted against days after drug removal. The error bar is ±1SD.
(B) DNA-damage responses of six individual p53 knockdown clones with more than 75% knockdown of p53. Expression of p53 on Western blot was first normalized to GAPDH, and relative level was then compared to mock knockdown. Survival rates of different clones upon DNA damage were analyzed as for pooled knockdown cells in (A). All numbered columns are p53 knockdown clones, and black column is mock knockdown. The error bar is +1SD.
(C) DNA-damage assay in p53 null MDA-MB-231 breast cancer cells was performed, as for MCF-7 cells. The survival rates of Ad/lacZ or Ad/HIC1 infected cells were normalized to mock-infected cells. The error bar is +1SD.
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4. Figure 3 HIC1 Interacts with SIRT1
(A) Illustration of important functional domains of HIC1. The central GLDLSKK motif recruits a HDAC1/CtBP repression complex, and the POZ domain interacts with unknown proteins (?) for repression. A putative nuclear localization signal (NLS) is located at the end of POZ.
(B) COS-7 cells were transfected with increasing amount of CMV promoter-driven mouse SIRT1 cDNA, followed by infection with Ad/HIC1. The 200 μg total lysate was immunoprecipitated with an HIC1 antibody, and one-fifth of the pulldown complex was loaded and blotted with a SIRT1 antibody. The input lane contained 10 μg total lysate (5%) from a 4 μg SIRT1 cDNA transfection, and about 16% of overexpressed SIRT1 was pulled down by HIC1 antibody.
(C) Coimmunoprecipitation of endogenous SIRT1 in nuclear extract of NIH 3T3 cells with an anti-HIC1 or control antibody. The blot was probed with anti-SIRT1, and one-half of the pulldown complex was loaded.
(D) Cotransfection of SIRT1 with 2 or 4 μg of HA-POZ fusion construct (triangle) or 4 μg HA in COS-7 cells. The total cell lysate was immunoprecipitated with an HA antibody, and one-fifth of each pulldown complex was loaded and blotted with anti-SIRT1.
(E) Effects of HIC1 POZ domain deletion. A mutant HIC1 was created by deleting the entire POZ domain (ΔPOZ) but retaining the NLS. MCF-7 cells were infected with Ad/lacZ, Ad/HIC1, or Ad/ΔPOZ and analyzed for DNA damage as in Figure 1D. Cell survival rates were all normalized to Ad/lacZ-infected cells. The error bar is +1SD.
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5. Figure 4 HIC1 Directly Regulates SIRT1 Transcription
(A) Mouse SIRT1 protein level in MEF nuclear extract from Hic1, LSH, or INK4a locus knockout. The numbers underneath Western blots are relative SIRT1 levels normalized to lamins.
(B) SIRT1 RNA levels in Hic1 knockout MEF by Northern blot. 18S RNA was used as a loading control. The numbers underneath the SIRT1 blot are relative SIRT1 levels normalized to 18S RNA.
(C) Overexpression of HIC1, but not lacZ or mutant HIC1, repressed nuclear SIRT1 in breast cancer MCF-7 cells.
(D) Luciferase reporter assay of SIRT1 promoter. A DNA fragment covering the entire SIRT1 promoter CpG island (from –1231 to +900) was isolated to drive luciferase expression in a pGL2 vector. The luciferase activity was assayed in COS-7 cells with expression of the constructs and infection with recombinant adenoviral vectors, as shown. The error bar is +1SD.
(E) ChIP assay with HIC1 and SIRT1 on the SIRT1 promoter. Two HIC1 binding sites (−1116 and −1039 in the same orientation), as indicated by wide arrows, are located 5′ to the promoter CpG island, and the other two (+575 and +660) in the opposite orientation to one another are located inside intron 1 toward the 3′ end of the island. ChIP was performed with HIC1 polyclonal antibody or SIRT1 monoclonal antibody, and both 5′ and 3′ binding regions were examined by multiplex PCR with GAPDH as an internal nonbinding control. CTL (R), normal rabbit IgG control; CTL (M), normal mouse IgG control. Triangles indicate increasing amount of HIC1 or SIRT1 antibodies.
(F) ChIP upon ChIP assay for 5′ HIC1 binding sites on SIRT1 promoter. Sonicated WI38 cell chromatin was first immunoprecipitated with rabbit HIC1 antibody, and the eluted product was reimmunoprecipitated with mouse SIRT1 antibody (HIC1, SIRT1). For control, normal rabbit IgG was used for the first round of immuoprecipitation and normal mouse IgG for the second (CTL [R, M]). ChIP upon ChIP was also performed with a reverse order of immunoprecipitation, namely, SIRT1 ChIP followed by HIC1 ChIP (SIRT1, HIC1) or control mouse IgG followed by rabbit IgG (CTL [M,R]). PCR amplification of 5′ HIC1 binding sites was carried out as in (E).
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6. Figure 5
Upregulation of SIRT1 in Primary Mouse Tumors with Loss of Hic1 Function
Tumors derived from Hic1+/− mice in (A), (B), (D), and (F) had loss of Hic1 expression, while the tumor in (E), normal lymph nodes (C), and lung airway (A and B) retained Hic1 expression (Chen et al., 2003). In (A), immunohistochemistry with SIRT1 antibody on a pulmonary adenocarcinoma is shown. Notice the intense SIRT1 staining (brown) in the tumor (green arrow), compared to the low level of SIRT1 in the adjacent normal airway (red arrow). The section encompassed by the square is enlarged in (B). In (C) and (D), high SIRT1 expression was found in a lymphoma (D) but not in the adjacent normal lymph node (C). In (E) and (F), higher SIRT1 expression was seen in a soft tissue sarcoma with loss of Hic1 (F) than in a sarcoma retaining Hic1 (E).
Cell  , DOI: ( /j.cell )
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7. Figure 6
SIRT1 Is the In Vivo Mediator of HIC1-Controlled Apoptotic DNA-Damage Responses
(A) Analysis of effects of dominant-negative SIRT1 mutants. Hic1−/− MEF were transfected with pBabe Sir2α H355A, pBabe SIRT1 H363Y, or empty pBabe vector and then treated with 40 μM etoposide. Survival cell numbers were measured 3 days after etoposide removal. The error bar is ±1SD.
(B) Analysis of p53 acetylation. MCF-7 cells were infected for 24 hr and treated with etoposide overnight, as indicated. The total cell lysates were precipitated with p53 antibody DO-1 and analyzed by Western blot with a specific antibody to acetylated p53 lysine 382. The membrane was then stripped and analyzed for total p53 with a rabbit polyclonal antibody. Equal amounts of total lysates were examined for GAPDH on a separate blot as an input loading control. Relative levels of acetylated and total p53 were normalized to GAPDH.
(C) Effects of TSA on p53 acetylation. MCF-7 cells were infected overnight with Ad/HIC1, and after removal of the virus, cells were treated with 0, 30, or 300 nM TSA for 10 hr, a time window for maximal global histone acetylation (Chen and Townes, 2000). Etoposide was then added to cells to final concentration of 20 μM without removing TSA, and cells were cultured for an additional 16 hr. Nuclear extracts were then prepared for Western blot analysis for p53 lysine 382 acetylation. Relative levels of p53 acetylation were normalized to lamins and indicated underneath the p53 blot.
(D and E) Analysis of expression changes of apoptotic target genes of p53 in MCF-7 cell lysates with infection of recombinant viruses and drug treatment as indicated. Relative levels of BCL-2, Bax, and Noxa were normalized to GAPDH and given underneath each Western blot.
(F) Effects of HIC1 knockdown. WI38 cells were mock-transfected or transfected with 12.5 nM siRNA for GAPDH or HIC1 for 24 hr. Transfection medium was removed, and cells were allowed to recover for 24 hr and were then treated with 20 μM etoposide for 12 hr. Nuclear extracts were prepared for Western blot analysis. Normalized relative levels of HCI1, total p53, and acetylated p53 are indicated underneath each Western blot, and SIRT1 levels are indicated on the top of its blot.
Cell  , DOI: ( /j.cell )
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8. Figure 7 A Model for the Roles of HIC1 in Tumor Suppression
A circular regulation of HIC1, SIRT1, and p53 is proposed for modulation of cellular responses to DNA damage. HIC1 represses the transcription of SIRT1, SIRT1 deacetylates p53 posttranscriptionally, and p53 trans-activates HIC1. The details are discussed in the text.
Cell  , DOI: ( /j.cell )
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