1. Volume 11, Issue 5, Pages 1265-1277 (May 2003)
MOF-Regulated Acetylation of MSL-3 in the Drosophila Dosage Compensation Complex 
Alessia Buscaino, Thomas Köcher, Jop H Kind, Herbert Holz, Mikko Taipale, Kerstin Wagner, Matthias Wilm, Asifa Akhtar 
Molecular Cell 
Volume 11, Issue 5, Pages (May 2003)
DOI: /S (03)

2. Figure 1
Double-Stranded RNA Interference of Dosage Compensation Proteins
(A) SL-2 cells were incubated with MSL-2 dsRNA (for 4 days) or MSL-3 or MOF dsRNA (for 2 days). Whole-cell extracts (WCE) were prepared from control cells (lanes 1–3) as well as dsRNA-treated cells (lanes 4–12), and the proteins were separated by SDS-PAGE and probed with antibodies against MSL-2, MSL-3, MLE, MOF, and tubulin. In order to assess the extent of protein reduction by dsRNA treatment, protein extracts were titrated as indicated. The asterisk shows that for this particular blot, MSL-3 dsRNA-treated extract corresponding to 100% of the extract was loaded and probed with MSL-2 antibody.
(B) Control and dsRNA-treated cells were fixed and immunostained with MSL-1, MSL-2, MOF, MSL-3, and MLE antibody or Hoechst as indicated.
(C) Quantitation of the X chromosomal localization of dosage compensation proteins upon treatment with dsRNA. In each experiment, 200 cells were counted and scored for localization to the X chromosome in three independent experiments, where cells were treated with dsRNA for MSL-2, MSL-3, and MOF, respectively. The bars show the percentage localization observed in each immunostaining, with standard error of mean represented on top of each bar. Black bars, immunostaining in control cells; whitebars, immunostaining of MSL-2 dsRNA-treated cells; light gray bars, immunostaining of MSL-3 dsRNA-treated cells; and dark gray bars, immunostaining of MOF dsRNA-treated cells.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 MSL-3 Association to the X Chromosome Is RNase Sensitive
(A) SL-2 cells were permeabilized with detergent (AA–AL) and subsequently untreated (AB, AF, and AJ) or treated with RNase A (AD, AH, and AL) for 10 min; this was followed by fixation and immunostaining with antibodies against MSL-3 (AB and AD), MSL-1 (AF and AH), and MOF (AJ and AL). Nuclei in (AA), (AC), (AE), (AG), (AI), and (AK) were stained with Hoechst
(B) Top, whole-cell extracts were prepared and immunoprecipitated with either preimmune serum (lanes 1 and 3), control beads (5), or MSL-3 antibody (lane 2), MOF antibody (lane 4), or MLE antibody (lane 6). Bottom, RNA was extracted from the samples indicated above followed by RT-PCR analysis. 25 PCR cycles were performed with roX2 or U6 primers. IP, immunoprecipitated sample; SN, supernatant. The position of the band corresponding to IgG is indicated by an asterisk.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 MOF Acetylates MSL-3
(A) MOF and MSL-3 interact directly. Recombinant MSL-3 was bound to MSL-3 antibody immobilized on protein agarose beads (lane 1). Following binding, beads were washed and incubated with MOF protein (lane 1 and lane 2). As a control, MSL-3 antibody immobilized on protein agarose beads was incubated with MOF in the absence of prebound MSL-3 (lane 2). Lane 3 MOF input, 50%. The membrane was first probed with MSL-3 antibody (top) and subsequently stripped and reprobed with MOF antibody (bottom).
(B) MOF protein was incubated with either MSL-3 (lanes 1 and 3) or alone with 3H-labeled acetyl CoA (lanes 2 and 4). Lanes 1 and 2 show Coomassie-stained gel; lanes 3 and 4 show the corresponding autoradiograph of the same gel. Relative mobility of the MOF and MSL-3 is indicated. Degradation product of MOF is indicated by an asterisk.
(C) SL-2 cells were treated with 200 ng/ml of TSA and 0.2 mCi/ml 3H-labeled acetic acid and incubated overnight at 25°C. The whole-cell extracts were prepared from these cells and incubated without (lane 1) or with (lane 2) MSL-3 antibody. Following incubation with agarose beads and subsequent washing, the samples analyzed by Western blot analysis using MSL-3 antibody (top panel). Lanes 1 and 2 correspond to control and immunoprecipitated samples (IP); lanes 3 and 4 correspond to 10% of supernatant (SN) of the corresponding samples. The acetylation status of MSL-3 was detected by autoradiography (bottom panel). The position of the band corresponding to IgG is indicated by an asterisk.
(D) SL-2 cells were either mock transfected (lanes 2 and 4) or transfected with FLAG epitope-tagged MSL-3 construct (lanes 1 and 3). 24 hr after transfection, cells were treated with 200 ng/ml of TSA and 0.2 mCi/ml 3H-labeled acetic acid and incubated overnight at 25°C. The whole-cell extracts were prepared from these cells and incubated with FLAG affinity beads. Following incubation, beads were washed and the samples analyzed by Western blot analysis using MSL-3 antibody (lanes 1 and 2). The acetylation status of MSL-3 was detected by autoradiography (lanes 3 and 4).
(E) SL-2 cells were either mock transfected (lane 3) or transfected with FLAG-tagged MSL-3 construct (lanes 1 and 2). Cells in lane 1 were also incubated with MOF dsRNA. 72 hr posttransfection cells were treated with 200 ng/ml of TSA and 0.2 mCi/ml 3H-labeled acetic acid and incubated overnight at 25°C. The whole-cell extracts were prepared and incubated with FLAG affinity beads. Subsequently, the samples were analyzed by Western blot analysis using FLAG antibody (bottom panel). The acetylation status of MSL-3 was detected by autoradiography (top panel).
(F) Precursor ion scan for the reporter ion m/z corresponding to the ammonium ion of acetylated lysine after the loss of ammonia. The spectrum of the analysis of the digest of the truncated protein is shown with the different charge states of the acetylated peptide with the mass of Da indicated.
(G) Determination of the acetylation site in the tryptic peptide with tandem mass spectrometry. The fragment spectrum of the peptide GGKAAHVEEPIVVPMDTGHLEAEHEMAPTPR is shown. The acetylated peptide was detected on a triple quadrupole mass spectrometer with precursor ion scanning for the reporter ion m/z 126.1, which is derived from an ammonium ion of acetylated lysine. The four times-charged precursor ion (m/z ) was chosen for fragmentation using a Q-TOF hybrid instrument. The N-terminal b-ion series identified the peptide and the acetylation site within the peptide. Additional singly charged y-ions and doubly charged ions (not assigned) confirmed the identification. The position of the acetylated lysine and the amino acids of the b-ion series are indicated.
(H) Point mutation in MSL-3 K116 residue abolishes MSL-3 protein acetylation by MOF. Schematic representation of MSL-3 protein, showing the position of the two chromodomains and the sites of mutagenesis. Mutant derivatives MSL-3 K G (lane 1), K116R (lane 2), and K116G (lane 3) were incubated with recombinant MOF and 3H-labeled acetyl CoA. The top panel shows the autoradiograph, while bottom panel shows the amounts of MSL-3 derivatives added as detected by Western blot analysis using MSL-3 antiserum. Degradation product of MOF is indicated by an asterisk.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
MSL-3 Localization to the X Chromosome Is Acetylation Sensitive
(A) SL-2 cells were permeabilized with detergent (AA–AJ) and subsequently untreated (AB) or treated with TSA for 10 min (AD) or 30 min (AF, AH, and AJ). This was followed by fixation and immunostaining with antibodies against MSL-3 (AB, AD, and AF), MSL-1 (AH), and MOF (AJ). Nuclei in (AA), (AC), (AE), (AG), and (AI) were stained with Hoechst
(B) Upper panel, whole-cell extracts were prepared from SL-2 cells that were permeabilized with detergent (lanes 1–4) and subsequently untreated (lanes 1 and 2) or treated with TSA for 30 min (lane 3). Extracts were then incubated with MSL-3 antibody (lanes 2 and 3) and half of the samples were analyzed by Western blot analysis. Lane 4 shows 10% of the input extract. The membrane was first probed with MSL-3 antibody (top) and subsequently stripped and reprobed with MOF antibody (bottom). Lower panel, the other half of the above samples was used for RNA extraction followed by RT-PCR analysis. For PCR using RoX2 and U6 primers, 24 or 28 cycles were performed. Lane 4 corresponds to the roX2 or U6 signal in control extract.
(C) Upper panel, whole-cell extracts were prepared from SL-2 cells that were permeabilized with detergent (lanes 1–4) and subsequently untreated (lanes 1 and 2) or treated with TSA for 30 min (lane 3). Extracts were then incubated with MLE antibody (lanes 2 and 3) and half of the samples were analyzed by Western blot analysis. Lane 4 shows 10% of the input extract. The membrane was first probed with MLE antibody (top) and subsequently stripped and reprobed with MOF antibody (middle) and finally MSL-3 antibody (bottom). Lower panel, the other half of the above samples was used for RNA extraction followed by RT-PCR analysis. 25 cycles were preformed for roX2 and U6 primers. Lane 4 corresponds to the corresponding roX2 or U6 signal in control extract.
(D) Upper panel, unacetylated (top) or acetylated (bottom) MSL-3 was incubated with paramagnetic beads alone (lane 1), paramagnetic beads bound to biotinylated Y14 DNA (lane 2), biotinylated Y14 RNA (lane 3), or biotinylated roX2 RNA (lane 4). Following binding, beads were washed and the bound fraction was separated on SDS PAGE, followed by Western blot analysis using MSL-3 antibody. Amount of bound MSL-3 is quantified by titration of the input MSL-3 as shown in the right panel. Quantitation of binding to each substrate is indicated as percentage of total protein input.
(E) Biotinylated roX2 RNA was bound to paramagnetic beads and nonacetylated (−) or acetylated (+) MSL-3 derivatives MSL3 K G (lanes 1 and 2) and MSL-3 K116G (lanes 3 and 4) were tested for RNA interaction. Lanes 4–8 show 10% supernatant (SN) loaded respectively. Degradation product of MSL-3 is indicated by an asterisk.
(F) Chromatinized (lanes 1 and 2) or free DNA (lanes 3 and 4) bound to paramagnetic beads was incubated with MSL-3 (lanes 1 and 3) or acetylated MSL-3 (lanes 2 and 4). Lanes 5 and 6 show 10% MSL-3 of the supernatant (SN) corresponding to lanes 1–4. Degradation product of MSL-3 is indicated by an asterisk. Quantitation of binding to each substrate is indicated as percentage of total protein input.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 RPD3 Association with the Dosage Compensation Proteins
(A) Drosophila nuclear extracts were used for immunoprecipitation using MSL-3 antibody (lanes 2 and 7) or preimmune serum (lanes 3 and 8) using binding buffer containing either 100 mM KCl (lanes 1–5) or 150 mM KCl (lanes 6–10). Lanes 4 and 5, and 9 and 10 show 10% of corresponding supernatants. Lanes 1 (I) shows 10% of extract used for the experiment. The samples were analyzed by Western blot analysis. The blot was first probed with MSL-3 and subsequently stripped with antibodies as indicated. The position of the band corresponding to IgG is indicated by an asterisk.
(B) Direct interaction between MSL-3 and RPD3. Recombinant MSL-3 was bound to protein agarose beads with (lane 1) or without (lane 3) MSL-3 antibody. This was followed by incubation with recombinant RPD3 in a binding buffer containing 100 mM KCl. RPD3 was also incubated with MSL-3 antibody bound to protein agarose beads (lane 2). Lane 4 shows 20% input (I) of RPD3.
(C) Deacetylation of MSL-3 with RPD3. MSL-3 fused to intein or intein alone was bound to chitin beads and acetylated with MOF (bars 1 and 3). Following removal of MOF and excess acetyl CoA, beads were incubate with RPD3 IP or control IP (compare bars 1 and 2 with 3 and 4). The bars show deacetylase activity with standard error of mean represented on top of each bar.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
Proposed Model
Dosage compensation can be viewed as a two-step process. The first step includes the assembly of the dosage compensation proteins, in particular MSL-1 and MSL-2, at the chromatin entry sites, followed by the recruitment of MLE, roX2, roX1, MSL-3, and MOF. This leads to assembly of a functional complex that is competent to spread from the entry sites along the length of the X chromosome. Regulated acetylation of MSL-3 may cause a conformational change that leads to temporary loss of its interaction with RNA. A cycle of deacetylation may follow that will allow MSL-3 to contact RNA again on a nearby affinity site. Through a continuous cycle of acetylation and deacetylation, MSL-3 along with the rest of the complex may be able to spread from a chromatin entry site. Chromatin entry sites are indicated as dark gray boxes, affinity sites as light gray boxes. HDAC, histone deacetylase; Ac, acetylated residues. A symbol for each DCC component is indicated.
Molecular Cell  , DOI: ( /S (03) )