1. Volume 26, Issue 1, Pages 103-115 (April 2007)
Heterochromatin Formation in Drosophila Is Initiated through Active Removal of H3K4 Methylation by the LSD1 Homolog SU(VAR)3-3 
Thomas Rudolph, Masato Yonezawa, Sandro Lein, Kathleen Heidrich, Stefan Kubicek, Christiane Schäfer, Sameer Phalke, Matthias Walther, Andreas Schmidt, Thomas Jenuwein, Gunter Reuter 
Molecular Cell 
Volume 26, Issue 1, Pages (April 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
The Drosophila Homolog of Histone Demethylase LSD1 Is the PEV Suppressor SU(VAR)3-3
(A) Protein structure of SU(VAR)3-3 and positions of the molecularly characterized mutations. SU(VAR)3-3 shows high identity with the human LSD1 protein, with 71% in the SWIRM and 75.3% in the amine oxidase domain. Yellow lozenge indicates the FAD binding domain.
(B) SU(VAR)3-3 is required for heterochromatic gene silencing. All Su(var)3-3 mutations were isolated as dominant suppressors of white variegation in wm4. They also suppress gene silencing in other PEV rearrangements (brown, Stubble, yellow, lacZ).
(C) Genomic extra copies of Su(var)3-9, Su(var)2-5, and Su(var)3-7 display enhanced silencing of the white gene in In(1)wm4 (upper row), which is impaired in the presence of the strong PEV suppressor mutant Su(var)3-3 (lower row). Rescue of the dominant suppressor effect of Su(var)3-3 mutations is found in wm4 flies heterozygous for a Su(var)3-3 mutation and a duplication (second column).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 SU(VAR)3-3 Demethylates In Vitro Mono- and Dimethyl H3-K4
Western blot analysis with α-mono-, α-di-, and α-trimethyl H3K4; α-mono- and α-dimethyl H3K9; and α-H3, α-HA, α-LSD1, and α-SU(VAR)3-3.
(A) Histone demethylation assays on bulk histones without enzyme and with mLSD1, mLSD K612A, SU(VAR)3-3, and SU(VAR)3-3 G316R recombinant protein.
(B) Western blot analysis of serial dilutions of the reaction products from HDM assays on bulk histones with SU(VAR)3-3 wild-type and G316R mutant protein. About 80% of H3K4me1 and H3K4me2 is removed from bulk histones upon incubation with SU(VAR) corresponds to 4.5 μl of reaction products.
(C) Concentration-dependent H3K4 demethylation activity of SU(VAR)3-3 on bulk histones. 1x corresponds to 2 μg of SU(VAR)3-3 protein.
(D) Mass spectrometric analyses of H3K4me1, -me2, and -me3 and H3K9me1 and -me2 levels in synthetic H3 peptides after incubation without enzyme and with mLSD1, mLSD K612A, SU(VAR)3-3, and SU(VAR)3-3 G316R.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
SU(VAR)3-3 Is a H3K4 Demethylase that Shows Ectopic Effects on In Vivo H3K9 Methylation
(A) Salivary gland polytene chromosomes from wild-type larvae stained with α-SU(VAR)3-3, α-dimethyl H3K4, and α-mono-, α-di-, or α-trimethyl H3K9.
(B) Histone methylation marks in Su(var)3-3 null polytene chromosomes. A loss of SU(VAR)3-3 causes chromosomewide increase in H3K4me2 and decreases the amount of H3K9 di- and trimethylation in chromocenter heterochromatin. Immunostaining with α-SU(VAR)3-3, α-dimethyl H3K4, and α-mono-, α-di-, or α-trimethyl H3K9.
(C) Ectopic effect on histone methylation after SU(VAR)3-3EGFP overexpression using a GAL4-SGS3 driver element results in heterochromatin association of SU(VAR)3-3EGFP, strong reduction of H3K4me2, and H3K9me2, and -me3. H3K9 monomethylation is not affected. Immunolabeling with α-SU(VAR)3-3, α-dimethyl H3K4, and α-mono-, α-di-, or α-trimethyl H3K9. DNA was stained with DAPI (red). Arrows point to chromocenter region.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Chromatin Association of SU(VAR)3-3 and Distribution of Histone Modification Marks at Cleavage and during Development of Somatic and Germline Precursor Cells
(A) During cleavage, SU(VAR)3-3 shows a uniform chromatin association until cycle 12. H3K4 dimethylation first becomes visible at the end of cleavage (cycle 12) and is excluded from the heterochromatic compartment and pole cells, the primordial germ cells of Drosophila. At cycle 14, SU(VAR)3-3 becomes enriched at prospective heterochromatin and remains uniformly associated with pole cell chromatin. H3K4 dimethylation is excluded from heterochromatin and pole cell chromatin.
(B) Redistribution of SU(VAR)3-3 during cleavage mitosis. The protein becomes accumulated at the chromosomal periphery during metaphase, whereas only weak uniform staining is detected during anaphase.
(C) H3K4me2, H3K9me2, and H3K9me3 staining in wild-type embryos and embryos produced by Su(var)3-3/+ females. Increase of H3K4me2 in blastoderm cells and significant H3K4me2 staining in pole cells of mutant embryos. Concomitant reduction of heterochromatic H3K9 di- and trimethylation in somatic and pole cell nuclei is observed. Arrowheads point to pole cells.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
High-Resolution Analysis of SU(VAR)3-3, HP1, and RPD3 Binding and Histone Modifications in Early Blastoderm Cells
(A) Immunostaining of wild-type (left panel) and embryos of Su(var)3-3−/+ females (right panel) with α-SU(VAR)3-3, α-HP1, α-RPD3, and α-acetyl H3K9. SU(VAR)3-3 distribution is shown for cycles 13–15 as indicated. All other photos represent cycle 14 embryos. In wild-type embryos, SU(VAR)3-3 is uniformly distributed during cycle 13, becomes enriched in heterochromatic regions during cycle 14, and afterwards is accumulated at the boundary region between eu- and heterochromatin (cycle 15). SU(VAR)3-3 and HP1 staining is significantly reduced in mutant embryos. RPD3 binding is almost uniform along the apicobasel axis of blastoderm nuclei in both wild-type and mutant embryos. In contrast, extended acetyl H3K9 staining toward heterochromatin is found in mutant embryos.
(B) Compared to wild-type (left panel) in Su(var)3-3 mutant embryos (right panel), the euchromatic mark H3K4me2 is elevated and spreads into the heterochromatic compartment. In mutant embryos, the heterochromatic trimethyl H3K9 is significantly reduced. No effects are found on mono- and trimethyl H3K4 and monomethyl H3K9. H3K9me2 appears only weakly reduced in Su(var)3-3 mutant embryos.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6
SU(VAR)3-3 Abolishes the Inhibitory Effect of K4-Methylated H3 Substrates on SU(VAR)3-9 Lysine 9 Methylation Activity, Associates with SU(VAR)3-9/HP1 Heterochromatin Complexes, and Impairs Heterochromatin Spreading in PEV
(A) HMTase assay with SU(VAR)3-9 and H3K4-methylated peptides (1–20) after incubation with SU(VAR)3-3. The amount of incorporated label was measured by scintillation counting after 20 min (error bars indicate minimum and maximum values).
(B) Affinity purification of SU(VAR)3-3 complexes from early (0–3 hr) embryos using a covalently Sepharose A-coupled SU(VAR)3-3 antibody. Immunoblotting of copurified proteins with α-SU(VAR)3-3, α-SU(VAR)3-9, α-HP1, and α-RPD3.
(C) Su(var)3-3 mutations impair heterochromatin spreading. ChIP analyses along the white-roughest euchromatic region juxtaposed in wm4 to pericentric heterochromatin.
In wm4;+/+ flies, a gradient of H3K9me2 along the white-roughest region is found (gray bars), whereas no H3K9 dimethylation is detected over this region at its normal position in the X chromosome (white bars). In Su(var)3-9 null mutant flies, H3K9me2 is not induced (black bars), whereas in Su(var)3-3 null flies, spreading of H3K9me2 is blocked (gray shaded bars). Error bars indicate standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7
Coordinated Histone Demethylation and Methylation Define the Boundary between Eu- and Heterochromatin in Early Embryonic Development
SU(VAR)3-3, the Drosophila homolog of human LSD1, associates in early embryonic development with heterochromatin. Protection of heterochromatin against the active histone methylation marks H3K4me1 and -me2 by the H3K4 demethylase SU(VAR)3-3 is a prerequisite for heterochromatin formation and establishment of heterochromatic H3K9 methylation by SU(VAR)3-9. The H3K4 demethylase SU(VAR)3-3 associates with the SU(VAR)3-9 complex for coordinated function in determination of the boundary between heterochromatic and euchromatic chromatin domains.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions