1. The Activation Potential of MOF Is Constrained for Dosage Compensation
Matthias Prestel, Christian Feller, Tobias Straub, Heike Mitlöhner, Peter B. Becker 
Molecular Cell 
Volume 38, Issue 6, Pages (June 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1 A Model System to Study the Sex-Specific Regulation by MOF
(A) MOF expression construct. Genomic DNA containing the mof locus and flanking sequences were modified by inserting a GAL4 DNA-binding domain and a FLAG tag. GAL4-DBD, GAL4 DNA-binding domain; CD, chromodomain; ZN, zinc finger domain; HAT, histone acetyltransferase domain.
(B) Reporter constructs (RC). The RC5 reporter has five binding sites for the Gal4 activator (5×UAS) located 5′ to the lacZ gene, which is controlled by the minimal hsp70 promoter. The transformation marker mini-white is transcribed from its own promoter. Arrows indicate the direction of transcription. In the RC3 construct the lacZ gene was inverted, such that the UAS sites reside 3′ of the lacZ gene.
(C and D) MOF activates transcription of the lacZ (C) and mini-white (D) genes. Total RNA was isolated from male (black bars) or female (open bars) adult flies. lacZ or mini-white transcripts were determined by RT-PCR and plotted as enrichments over an internal gapdh1 control value. The enrichments indicated above the bars refer to the RNA values in the presence of MOF-Gal4 relative to the values in its absence (panels to the left). Error bars represent mean ± SEM. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Chromatin Interactions upon MOF Recruitment
Chromatin constituents were monitored by ChIP in sorted male and female adult flies. The presence of sequences in the immunoprecipitate corresponding to four amplicons along the reporter loci (arrows, see schematics on top) was determined by qPCR. The RC3 locus was probed in (A), (C), and (E) and the RC5 locus in (B) and (D). Chromatin was from females or males of the reporter lines in the absence of MOF-Gal4 (light and dark gray bars, respectively) or in the presence of MOF-Gal4 (white and black bars). Error bars represent mean ± SEM. (A) and (B) display MOF interactions at the RC3 and RC5 reporters, respectively. (C) and (D) show the corresponding H4K16 acetylation levels. (E) MSL2 interactions were revealed at the RC3 reporter. (F) Genome-wide correlation of gene expression and H4K16ac levels in SL2 cells, using the values of Kind et al. (2008). (G) Binding of the polymerase II subunit Rpb3 5′ to the lacZ gene. All ChIP experiments were internally controlled by monitoring interactions at the X chromosomal and dosage compensated armadillo gene. Error bars represent mean ± SEM. See also Figure S2.
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4. Figure 3 MBD-R2 Binds Active Genes Globally
(A) Chromosome-wide binding of MBD-R2 in male flies. Oligonucleotide probe signals are plotted along the major chromosomes as indicated (note that the entire X chromosome is represented, but only parts of each autosome). Significant bound probes (tileHMM, see the Supplemental Experimental Procedures) are marked in red. Numbers along the x axis denote the physical position along the chromosomes in megabase pairs (Mb).
(B) MBD-R2 binds active genes independent of expression level. Genes were grouped in equal-sized bins according to their expression levels (GST RNAi expression set, see Figure 5). The ordinate depicts the signal distribution of average gene binding values in the respective groups, assessed by MBD-R2 ChIP-chip in SL2 cells.
(C) MBD-R2 binding coincides in male and female flies. Correlation of the ChIP-chip binding signal (averaged gene binding score, see the Supplemental Experimental Procedures) of X chromosomal genes (upper panel) or autosomes (lower panel) from at least three biological replicates from both sexes. Spearman correlation coefficient rho = 0.94 (p < 2.2e-16).
(D) ChIP-chip profiles of MBD-R2 in male and female flies along a representative X chromosomal region. The profiles are related to a gene representation at the bottom of the panel, where genes drawn above the line are transcribed from left to right and genes below the line are transcribed from right to left. Active genes are marked in red, inactive genes in black (Chintapalli et al., 2007). The x and y axis are denoted as in (A).
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
MBD-R2 and MOF Colocalize on Active Genes under All Circumstances, Except for the Male X Chromosome
(A and B) Correlation between chromosomal binding of MOF and MSL1 or MOF and MBD-R2 on male (A) or female (B) chromosomes. Scatter plots of averaged binding score per gene (see the Supplemental Experimental Procedures) of MBD-R2, MOF, and MSL1 on male chromosomes as indicated. All spearman correlation coefficients (rho) are p < 2.2e-16.
(C) The majority of active genes on the male X are bound by MBD-R2, MOF, and MSL1. Venn diagram of significantly bound active genes.
(D) Binding of MSL1, MOF, and MBD-R2 along a representative region of the X chromosome in males (left panel) and females (right panel). The profiles are related to the gene representation at the bottom of the panel. All genes are active. Genes depicted above or below the line of physical coordinates are transcribed from left to right or right to left, respectively.
(E) Differential distribution of MOF and MBD-R2 across the gene bodies. Cumulative plots of MOF (left) and MBD-R2 (right). ChIP-binding signals along genes were aligned at the transcriptional start site (TSS) and transcriptional termination site (TT). The average binding score is plotted 2 kb bp up- and downstream of the TSS and TT in a sliding window (10 bp step size, 300 bp window). For this analysis, 637 X chromosomal genes and 740 autosomal genes were selected, which are minimally 2 kb long, do not overlap with other genes, and are expressed. The genes were grouped according to their gene expression level (Chintapalli et al., 2007) into “low” (black), “intermediate” (orange), “high” (blue), and “very high” (purple) categories. The x axis denotes the distance to the TSS and TT in base pairs.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 MBD-R2 Is a Genome-wide Transcriptional Activator
Active genes (Muse et al., 2007) were grouped according to their averaged MBD-R2 binding score and plotted against their expression change after MBD-R2 RNAi (log2 [MBD-R2 RNAi/control RNAi]). Genes were categorized in groups with increasing binding score as represented in the box plots. Genes were considered “active” if elongating polymerase was bound significantly in the ChIP-chip profile of Muse et al. (2007). Similar results were obtained if genes with an expression value of at least 4 in the control samples (Affymetrix scale) were classified as “active.”
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Sex-Specific Cofactor Recruitment upon MOF Binding
(A) Schematic illustration of the relative occupancy of chromatin constituents at the 5′ lacZ site in male and females. The graph summarizes data presented in Figures 2B, 2D, and 6B and data not shown for MSL2. Displayed are the ratios of ChIP enrichment of the indicated antibodies in females (white bar) and males (black bar). MOF or H4K16ac levels are similar in both sexes. MSL2 is only enriched in males (background levels in females). Females lacking MSL2 accumulate more MBD-R2 than males.
(B and C) Ectopic expression of MSL2 (B) and MSL1 (C) in females leads to enrichment of the corresponding protein at the RC5 locus upon MOF recruitment (control represents the RC5 locus in the absence of MOF-Gal4). Error bars represent mean ± SEM. Overexpressing MSL1 males could not be analyzed due to lethality. White bars, female flies; black bar, male flies.
(D) ChIP with MBD-R2 in females expressing either MSL2 as in (B) or MSL1 (C). Tethering MOF to the lacZ reporter leads to a 2-fold enrichment of MBD-R2 in females versus males. Expression of MSL2 (pseudomales) reduces the MBD-R2 levels. Expression of MSL1 has only a minor impact. Presentation as in (B).
(E and F) Change of lacZ induction (β-galactosidase activity) by MOF upon ectopic expression of MSL2 (E) or MSL1 (F). Presentation as in (B).
(G) Reporter gene activity in SL2 cells after ablation of MSL2 and MBD-R2. SL2 cells were treated with dsRNA against GST (control), MSL2, or MBD-R2 sequences. Three days after RNAi treatment, cells were transfected with an expression vector for Gal4-MOF and a firefly luciferase reporter furnished with UASGal elements either 5′ of the minimal thymidine kinase promoter or 3′ of the firefly luciferase gene. Transfection efficiency was normalized by coexpression of a Renilla luciferase expression plasmid.T test, two-sided and unpaired, error bars represent mean ± SEM.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
MOF Redistributes to the X Chromosome in Female Flies Overexpressing MSL2
(A and B) Box plots present ChIP-chip signals of autosomal or X chromosomal probes, which represent the binding of MOF (A) and MBD-R2 (B) in wild-type (WT) females, MSL2 expressing females (pseudomales) and wild-type males. The ordinates depict the log2-normalized values of probes. The 99% confidence interval (CI) of the true difference in means between X chromosomal and autosomal MOF binding is shown. The red line depicts the median from all probes.
(C) Chromosome-wide difference plots of MOF binding between pseudomales and females. The difference of oligonucleotide probe signals between pseudomales and females is plotted along the major chromosomes as indicated (note that only part of the autosomes are represented). Significant (adjusted p values < 0.05) gains are marked in red, significant losses are marked in blue. Numbers along the x axis denote the physical position along the chromosomes in megabase pairs (Mb).
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions