1. Volume 20, Issue 4, Pages 601-611 (November 2005)
Monoubiquitination of Human Histone H2B: The Factors Involved and Their Roles in HOX Gene Regulation 
Bing Zhu, Yong Zheng, Anh-Dung Pham, Subhrangsu S. Mandal, Hediye Erdjument-Bromage, Paul Tempst, Danny Reinberg 
Molecular Cell 
Volume 20, Issue 4, Pages (November 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
Partial Purification of a Histone H2B-K120 Monoubiquitination Activity from HeLa Extracts and the Purification of Human RNF20/40 Complex
(A) The purification scheme for a partially purified histone H2B-K120 monoubiquitination activity.
(B) The monoubiquitination activity is specific for histone H2B-K120.
(C) The histone H2B-K120 monoubiquitination activity is nucleosome specific.
(D) The histone H2B-K120 monoubiquitination activity is a ∼600 kDa complex. Fractions were from a 2 ml Smart Superose 6 gel filtration column. The fraction numbers are shown in the top of the panel.
(E) Silver stain of the affinity-purified human RNF20/40 complex. Fractions were from a 2 ml Smart Superose 6 gel filtration column. The fraction numbers are shown at the top of the panel. The subunits of the human RNF20/40 complex are indicated at the right side.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
UbcH6 Is the E2-Conjugating Enzyme, and RNF20/40 Complex Is the E3 Ligase for Histone H2B-K120 Monoubiquitination
(A) UbcH6 and RNF20/40 specifically monoubiquitinate the nucleosome substrate.
(B) The activity mediated by UbcH6 and RNF20/40 complex is specific for histone H2B-K120.
(C) The activity mediated by UbcH6 and RNF20/40 complex is specific for nucleosome substrate.
(D) His-tagged UbcH6 specifically pulls down the purified RNF20/40 complex in a nickel-agarose pull-down assay.
(E) Antibody against FLAG immunoprecipitates UbcH6 in the presence of purified FLAG-RNF20/40 complex.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Trimeric Association of the PAF Complex, UbcH6, and RNF20/40 Complex Stimulates Histone H2B-K120 Monoubiquitination In Vitro
(A) All the subunits of the PAF complex are present in the native partially purified histone H2B-K120 monoubiquitination activity assayed in Figures 1B and 1C.
(B) Westerns indicating the immunodepletion of the PAF complex from the native partially purified histone H2B-K120 monoubiquitination activity.
(C) Depletion of PAF complex from the native partially purified histone H2B-K120 monoubiquitination activity reduces the activity that was rescued upon supplementation with purified PAF complex.
(D) His-UbcH6 specifically pulls down the purified PAF complex in a nickel-agarose pull-down assay.
(E) Antibody against Paf1 immunoprecipitates UbcH6 in the presence of purified PAF complex.
(F) RNF20/40 complex and PAF complex interact only in the presence of UbcH6.
(G) Trimeric association of PAF complex, UbcH6, and RNF20/40 complex stimulates histone H2B-K120 monoubiquitination activity.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Human RNF20/40 Complex, UbcH6, and the PAF Complex Are Present at Transcriptionally Active Genes
(A) ChIP experiments showing RNF20/40 complex, UbcH6, and PAF complex are present at the constitutively expressed RPB1 gene.
(B) ChIP experiment showing the recruitment of RNF20/40 complex, UbcH6, and PAF complex together with the RNA Polymerase II transcription machinery to the MAGE-A1 gene upon its induction.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Overexpression of RNF20 Results in Elevated Histone H2B Monoubiquitination In Vivo
(A) Coomassie blue staining of hydroxyapatite-purified core histones.
(B) Histone H2B monoubiquitination levels are elevated upon overexpression of RNF20.
(C) Overexpression of RNF20 also leads to elevated levels of histone H3-K4 mono- and trimethylation and H3-K79 dimethylation.
(D) Quantification of (B); error bars represent the standard error of the mean.
(E) Knockdown of RNF20 and RNF40. RNAi targeting RNF20 and/or RNF40 verified by RT-PCR.
(F) The level of u-H2B, but not of u-H2A, is reduced upon RNAi against the RNF20/40 complex.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Perturbing the Level of Histone H2B Monoubiquitination Results in Misregulation of the Hox Genes
(A) Overexpression of RNF20 leads to elevated Hox gene expression.
(B) Quantification for selected genes tested in (A).
(C) RNAi targeting hSki8 or hCtr9 reduces Hox gene expression.
(D) Quantification of selected genes tested in (C).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Overexpression of RNF20 Leads to Elevated Levels of Histone H3-K4 Trimethylation and H3-K79 Dimethylation at the Hox Genes
Molecular Cell  , DOI: ( /j.molcel )
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