1. Volume 21, Issue 1, Pages 51-64 (January 2006)
ING Tumor Suppressor Proteins Are Critical Regulators of Chromatin Acetylation Required for Genome Expression and Perpetuation 
Yannick Doyon, Christelle Cayrou, Mukta Ullah, Anne-Julie Landry, Valérie Côté, William Selleck, William S. Lane, Song Tan, Xiang-Jiao Yang, Jacques Côté 
Molecular Cell 
Volume 21, Issue 1, Pages (January 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Characteristic Features of the Human ING Family of Tumor Suppressor Proteins
(A) Sequence alignment of the five human ING proteins. Three domains, represented by black boxes, are conserved. Based on sequence comparison, the INGs were subdivided in three different groups (ING1/2 in red, ING3 in blue, and ING4/5 in green).
(B) TAP/FLAG-purified ING2 complex from HeLa S3 cells expressing FLAG-ING2-TAP was subjected to SDS-PAGE followed by silver staining (left panel). Interacting proteins identified by mass spectrometric analysis are indicated on the right (numbers of independent peptides obtained vary between three and 50 per indicated protein). Western blot analysis of the preparation shows the association of ING2 in a Brg1-HDAC1/2 complex.
(C) Silver staining of TAP-purified ING3 complex (left panel) and Western blot analysis using antibodies specific to Tip60 complex subunits. Asterisks indicate nonspecific protein bands.
Molecular Cell  , 51-64DOI: ( /j.molcel )
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3. Figure 2
The Purified ING4 Complex Contains the HBO1 Histone Acetyltransferase
(A) Silver staining of the ING4-TAP complex. Interacting proteins identified by mass spectrometric analysis are indicated on the right (between eight and 66 peptides per indicated protein).
(B) Schematic representation of the protein domains present in the identified subunits. JADE proteins are also known as PHF15/16/17.
(C) Western blot analysis of TAP purifications of ING4 and Tip60 that shows two distinct complexes.
(D) Fractions from Tip60- and ING4-TAP were tested for HAT activity specificity on human core histones, oligonucleosomes, or recombinant nucleosomes substrates.
(E) Reciprocal purification of HBO1 complex (by TAP/FLAG triple affinity) was subjected to SDS-PAGE followed by silver staining. Interacting proteins identified by mass spectrometric analysis are indicated on the right (between two and 12 peptides per indicated protein).
(F) Western blot analysis of purified HBO1-TAP.
(G) Purified HBO1-TAP was assayed for HAT activity on core histones and oligonucleosomes.
(H) Luciferase assays of p53-dependent transcription. Reporter construct containing the p21 promoter was cotransfected with the indicated expression vectors in RKO versus RKO-E6 cells and assayed for luciferase activity (standard deviations are based on three different experiments).
Molecular Cell  , 51-64DOI: ( /j.molcel )
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4. Figure 3
HBO1 Is the Major Histone H4 Histone Acetyltransferase In Vivo and Is Required for Cell Cycle Progression
(A) HAT specificity of purified ING4-TAP was tested on recombinant nucleosomes and analyzed by Western blot with the indicated antibodies.
(B) 293T cells, cotransfected with the indicated pSUPER plasmids and caax-GFP, were sorted 48 hr posttransfection and subjected to acidic extraction. Histones were analyzed by Western blot with the indicated antibodies.
(C) Cells prepared as in (B) but sorted 24 hr after transfection were subjected to MTT assay during 3 days after seeding to monitor growth.
(D) Flow cytometry indicated the DNA content at 72 hr posttransfection of the GFP-positive cells described in (B).
(E and F) MCF7 cells treated for 48 hr with the indicated pSUPER plasmids were marked with BrdU for 30 min and analyzed by flow cytometry for BrdU incorporation and (F) for analysis of DNA content to determined percentage of cells in S phase that incorporated BrdU (mean of three experiments).
(G) Subcellular localization of HBO1 and Tip60 was determined by indirect immunofluoresence and confocal microscopy on MCF7 cells. Nuclei were visualized with DRAQ5.
(H) Double staining for HBO1 (green) and Tip60 (red).
Molecular Cell  , 51-64DOI: ( /j.molcel )
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5. Figure 4
ING5 Associates with Three Different MYST Histone Acetytransferases: HBO1, MOZ, and MORF
(A) TAP-purified ING5 protein was subjected to SDS-PAGE and visualized by SYPRO ruby staining. Proteins identified by tandem mass spectrometry are indicated (between two and 50 peptides per protein).
(B) Schematic representation of protein domains present in the identified subunits.
(C) Western blot analysis of purified ING4- and ING5-TAP complexes with indicated antibodies.
(D) Purified ING5-TAP and ING4-TAP were tested for HAT activity on core histones or oligonucleosomes.
(E) Purified ING4- and ING5-TAP fractions were immunoprecipitated with the indicated antibodies, and bound material was analyzed by HAT assay on chromatin substrate.
(F) HAT specificity of ING3 and ING5 complexes was tested on recombinant nucleosomes with purified fractions and analyzed by Western blot with the indicated antibodies.
(G) A luciferase reporter construct containing six Runx2 binding sites was cotransfected with the indicated expression vectors, and cell extracts were analyzed for luciferase activity (mean of three different experiments).
Molecular Cell  , 51-64DOI: ( /j.molcel )
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6. Figure 5
The ING5 HAT Complexes Interact with the MCM Helicase and Are Essential for DNA Replication
(A) Purified ING4-TAP and ING5-TAP fractions were compared for presence of MCM proteins by Western blot with indicated antibodies.
(B) Purified HBO1-TAP was analyzed by Western blot with anti-MCM2.
(C) Flow cytometry analysis indicates the DNA content of 293T cells at 48 hr posttransfection with the indicated siRNA.
(D) Acid extracted histones from cells as in (C) were analyzed by Western blot.
(E–G) MCF7 cells treated for 48 hr with the indicated pSUPER plasmids were marked with BrdU for 30 min and analyzed by flow cytometry for BrdU incorporation and (F) analyzed for DNA content to determine the percentage of cells in S phase that incorporated BrdU (G) (mean of two experiments).
Molecular Cell  , 51-64DOI: ( /j.molcel )
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7. Figure 6
ING4 and ING5 Subunits Are Required for HBO1 to Acetylate Chromatin Substrates
HBO1 complexes from transduced HeLa S3 cells expressing FLAG-HBO1-TAP (or nontagged) and transfected with the indicated siRNAs were immunoprecipitated with anti-FLAG M2 resin, and bound material was analyzed for HBO1 recovery (anti-FLAG Western) (A), HAT activity on free histones (direct counts) (B), and ratio of HAT activity on oligonucleosomes versus free histones (results are presented relative to the siRNA control sample set to 1) (C).
Molecular Cell  , 51-64DOI: ( /j.molcel )
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8. Figure 7
Schematic Representation of the Different Histone Acetyltransferases and Deacetylases Complexes that Contain a Member of the Yeast or Human ING Family of Proteins
Molecular Cell  , 51-64DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions