The drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation - PubMed (original) (raw)

The drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation

E R Smith et al. Mol Cell Biol. 2000 Jan.

Abstract

In Drosophila, dosage compensation-the equalization of most X-linked gene products in males and females-is achieved by a twofold enhancement of the level of transcription of the X chromosome in males relative to each X chromosome in females. A complex consisting of at least five gene products preferentially binds the X chromosome at numerous sites in males and results in a significant increase in the presence of a specific histone isoform, histone 4 acetylated at lysine 16. Recently, RNA transcripts (roX1 and roX2) encoded by two different genes have also been found associated with the X chromosome in males. We have partially purified a complex containing MSL1, -2, and -3, MOF, MLE, and roX2 RNA and demonstrated that it exclusively acetylates H4 at lysine 16 on nucleosomal substrates. These results demonstrate that the MSL complex is responsible for the specific chromatin modification characteristic of the X chromosome in Drosophila males.

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Figures

FIG. 1

FIG. 1

MOF acetylates histone H4. A recombinant fragment of MOF was expressed in E. coli and assayed for acetyltransferase activity on free histones. MOF acetylates histones predominantly on histone H4 and to a lesser extent on histones H3 and H2A. For comparison, the activities of yeast acetyltransferases Gcn5p and MOF-related Esa1p are shown.

FIG. 2

FIG. 2

Epitope-tagged MSL2 colocalizes with MOF and histone H4 acetylated at lysine 16 (H4Ac16) on the X chromosome in S2 cells. (A) Cells were transfected with a construct expressing MSL2 fused to the HA epitope. Tagged MSL2 is detected with the 12CA5 monoclonal antibody, while endogenous MOF is detected with a rabbit polyclonal antibody to the C terminus of MOF. The top panel shows that a small fraction of the cells contain transfected MSL2-HA, which is seen colocalizing with endogenous MOF. The bottom panel shows an enlargement of a nucleus where both antibodies can be seen painting a chromosome that traverses the nucleus. (B) Stable cell lines were selected with hygromycin, and a line that expressed a low level of basal (uninduced) expression was chosen for further study. In this line, basal levels of MSL2-HA can be detected along the length of the X chromosome, coincident with H4Ac16 and the other MSLs (not shown). Occasional areas of nonoverlap may reflect the presence of partial or nonfunctional complexes.

FIG. 3

FIG. 3

Major immunoprecipitated proteins detected by silver staining correspond to known MSLs. The 12CA5 antibody (HA) generates the same set of proteins from MSL2-HA (M2HA) nuclear extracts that the MSL1 antiserum generates from S2 cell extracts; differences in stochiometry are ascribable to the overexpression of HA-tagged MSL2. This set of proteins is absent from HA or control immunoglobulin G (PI) precipitates from (untransfected) S2 cells. Western analysis of the MSL1 immunoprecipitate shows that the major silver-stained bands correspond to known MSLs. Rabbit anti-MSL1 serum detects a 170-kDa band, guinea pig anti-MSL2 detects a 135-kDa band, rabbit anti-MOF detects a 135-kDa band, and rabbit anti-MSL3 detects a 58-kDa doublet of bands. Molecular masses are indicated in kilodaltons (KDa).

FIG. 4

FIG. 4

MLE association with the MSL complex. Significant levels of MLE are detected with other MSLs when immunoprecipitations are performed under low-salt conditions (see Materials and Methods). MSL complexes were eluted from M2-Flag agarose and subjected to a second immunoprecipitation with anti-MSL1. (A) A silver-stained protein is visible between MSL1 and MSL2/MOF. Two concentrations (1× and 5×) of a stringently washed anti-MLE immunoprecipitate (IP) were loaded on the same gel; correlation between silver staining and Western staining intensities as well as comigration by SDS-PAGE, confirms that this band is MLE. (B) Comparison of low-salt (LS) and high-salt (HS) washing conditions reveals a salt-sensitive association of MLE with the other MSLs. As seen by Western analysis, significant levels of MLE are released from immunoprecipitates when incubated with high-salt buffers but not low-salt buffers; MSL1 levels are unaffected. A preimmune control (PI) was washed under low-salt conditions and reveals a low level of contaminating MSLs in these preparations.

FIG. 5

FIG. 5

roX2 is expressed in S2 cells and associates with the MSL proteins. (A) Northern analysis shows that both roX RNAs are expressed in adult Drosophila males (M) but not females (F); only roX2 was detected in S2 cells. The filters were reprobed for rp49 RNA as a loading control. (B) RNA was extracted from anti-MSL1 immunoprecipitates (M1) or the corresponding preimmune serum (PI) and subjected to RT-PCR. Agarose gel electrophoresis of PCR products and staining with ethidium bromide detected significant levels of roX2 RNA in the anti-MSL1 immunoprecipitates, while a lower level of roX2 RNA (1 to 2%) was detected in the preimmune serum. This level of contamination is consistent with the amount of contaminating MSL proteins observed in these immunoprecipitates (Fig. 4B). A kanamycin kinase transcript was used to monitor variation in processing of samples; comparable levels of this control RNA (Ctrl) were detected by RT-PCR in the preimmune and anti-MSL1 immunoprecipitates.

FIG. 6

FIG. 6

The MSL complex acetylates nucleosomal H4 in a MOF-dependent manner. (A) Immunoprecipitates were assayed for acetyltransferase activity toward mononucleosomes and processed for SDS-PAGE and fluorography. Both MSL1 immunoprecipitates (IP) from S2 cells and 12CA5 immunoprecipitates (HA) from MSL2-HA-expressing cells acetylate nucleosomes specifically on histone H4. Preimmune serum (PI) or 12CA5 monoclonal antibody does not immunoprecipitate any histone acetyltransferase activity from S2 cells. (B) Acetyltransferase activity was also assayed from cells transfected with the HA-tagged mof+ or HA-tagged mof1 allele (Gly-to-Glu mutation at residue 691). Western analysis demonstrates that the extracts contain complexes with large amounts of HA-tagged MOF or MOF1. Histone acetyltransferase activity from MOF1-containing complexes is dramatically reduced.

FIG. 7

FIG. 7

The MSL complex specifically acetylates lysine 16 of H4 on Drosophila nucleosomes. The inset shows a fluorogram of an acid-urea gel-separated H4 that has been acetylated by the MSL complex, in the presence of 3H-labeled acetyl-CoA and mononucleosomes. Most of the [3H]acetate is found on the monoacetylated band. A similar acid-urea gel was blotted to PVDF, and the monoacetylated band was cut out and subjected to microsequencing. The counts per minute released at each cycle are plotted against the residue sequenced. Essentially all of the counts are found at lysine 16, except for the sequencing lag that results in the release of residual counts at positions 17 to 20. Additionally, phenylthiohydentoin analysis detected acetyl-lysine only at position 16, not at positions 5, 8, and 12.

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