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Functional interplay between MSL1 and CDK7 controls RNA polymerase II Ser5 phosphorylation

Abstract

Proper gene expression requires coordinated interplay among transcriptional coactivators, transcription factors and the general transcription machinery. We report here that MSL1, a central component of the dosage compensation complex in Drosophila melanogaster and Drosophila virilis, displays evolutionarily conserved sex-independent binding to promoters. Genetic and biochemical analyses reveal a functional interaction of MSL1 with CDK7, a subunit of the Cdk-activating kinase (CAK) complex of the general transcription factor TFIIH. Importantly, MSL1 depletion leads to decreased phosphorylation of Ser5 of RNA polymerase II. In addition, we demonstrate that MSL1 is a phosphoprotein, and transgenic flies expressing MSL1 phosphomutants show mislocalization of the histone acetyltransferase MOF and histone H4 K16 acetylation, thus ultimately causing male lethality due to a failure of dosage compensation. We propose that, by virtue of its interaction with components of the general transcription machinery, MSL1 exists in different phosphorylation states, thereby modulating transcription in flies.

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Figure 1: Evolutionarily conserved binding of MSL1 to target promoters.
Figure 2: MSL1 depletion decreases Pol II Ser5 phosphorylation in flies.
Figure 3: MSL1 and CDK7 show genetic interaction, and CDK7 inhibition causes decreased MSL1 localization on the X chromosome.
Figure 4: MSL1-dependent CDK7 recruitment on chromatin.
Figure 5: MSL1 is a phosphoprotein in vivo and can be phosphorylated by CDK7 in vitro.
Figure 6: MSL1 phosphomutants show a decrease in Pol II Ser5p and do not rescue male viability.

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Acknowledgements

We thank all members of the Akhtar laboratory for helpful discussions. We especially thank K. Lam and B. Sheikh for critical reading of the manuscript and helpful suggestions. We would also like to thank K. Adelman (NIEHS), K. Johansen (Iowa State University), J. Kadonaga (UCSD), J.T. Lis (Cornell University) and B. Suter (University of Bern) for kindly providing antibodies. This work was supported by an EU-funded EpiGeneSys awarded to A.A. and N.M.L., and DFG-BIOSS II, CRC992, CRC1140 and CRC746, awarded to A.A. L.M.S. acknowledges funding from the DFG and NIH.

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Authors

Contributions

S.C. performed ChIP–seq for MSL1 D. virilis, ChIP–qPCR, RNA-seq, co-IP assays, immunofluorescence microscopy and WB analyses; H.H. purified baculovirus-expressed proteins and performed binding assays, kinase assays, WB analyses and antibody characterization; M.S. performed genetic crosses, ChIP–qPCR, antibody characterization and WB analyses; T. Chelmicki performed and analyzed mammalian ChIP–seq and ChIP–qPCR, and analyzed WB data; P.G. performed genetic crosses, analysis and quantification of the phenotypes; V.P., F.D., F.T.C., F.R., W.W., N.M.L. and L.M.S. performed bioinformatics analyses and contributed to the corresponding manuscript sections; T.M. guided the development and implementation of deepTools for NGS analysis and quality controls; P.D. performed evolutionary analysis of MSL1; T. Conrad and S.R. performed dmMSL1 ChIP–seq experiments; G.M. performed and analyzed the mass spectrometry data; A.A., S.C., M.S., T. Chelmicki, P.G. and V.P. designed experiments and analyzed the data; A.A., T. Chelmicki and M.S. prepared the manuscript.

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Correspondence to Asifa Akhtar.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Genome-wide binding analysis of MSL1 in different species.

(a) Related to Fig.1b. Co-immunostaining of D.melanogaster and D. virilis male larva polytene chromosomes with antibodies against MSL1 (green) and MOF (red). Here, dmMSL1 and dmMOF antibodies were used for immunostaining in both species.

(b) Related to Fig. 1a. Heatmaps show the input-normalized MSL1 ChIP-seq signals for X-linked target genes in male D. melanogaster and D. virilis samples. The signals were clustered individually, using k-means clustering (heatmapper tool of the deepTools suite59). This reveals two visibly distinct modes of MSL1 binding along the male X in D. melanogaster: one group of X-linked genes with broad gene body enrichment, one group of X-linked targets with promoter binding only. The MSL1 signal for the male X of D. virilis cannot be as readily subdivided as it consists of narrow, mostly promoter-proximal binding sites.

(c) Distribution of peak overlaps with promoter regions, gene bodies and intergenic regions. Promoters were defined as in Fig. 1a: 150 bp upstream of TSS in D. melanogaster and D. virilis, 1 kb upstream of TSS in M. musculus. Peaks overlapping with several features were counted only once.

(d) Table of peak counts for the different samples - related to the heatmaps in Fig. 1a as each number indicates the number of regions displayed in the individual parts of each heatmap.

Supplementary Figure 2 MSL1 depletion leads to decreased Pol II Ser5p in flies.

(a-c) Related to Fig. 2a-b. Depletion of MSL1, MSL2 and MOF in S2 cells. Note that MSL1 depletion affects MSL3, but not MCRS2, NSL3 and TBP levels. H3K4me3, an active mark at the promoters is unaffected. H2BK120ub, previously shown to be dependent on CDK712, is reduced upon MSL1 and MSL2 depletion. H4K16ac is severely affected in MSL1 and MSL2 knockdowns. Note that, depletion of MOF has no effect on Pol II Ser5p and Pol II Ser2p. 30% and 100% of the cell extracts were loaded. a-tubulin and histone H2B were used as loading controls.

(d) Ratio of Pol II Ser5p to Pol II RPB3 ChIP percentage recovery presented in Fig. 2f–2g.

(e) Input-normalized ChIP-seq signals for MSL1 and MOF in M. musculus for promoter-target genes and a randomly chosen subset (20%) of non-target genes. As in Fig. 1a, target genes were defined by an overlap of significant binding sites (peaks) for the respective ChIP sample with promoter regions (1kb upstream of TSS in M. musculus) of annotated genes. Heatmaps and summary plots were generated with the deepTools suite59. For target numbers and annotations, see also Supplementary Figures S1c-d.

(f-g) Depletions of MSL1 (f) and MSL2 (g) in mouse ES cells. 100%, 30%, 10% of respective dilution was loaded. GAPDH and histone H3 were used as loading controls.

Supplementary Figure 3 Genetic analysis of msl-1 mutants in flies.

(a) % viability of heterozygous vs homozygous females (top panel) and males (bottom panel) for three different msl-1 loss-of-function alleles (msl1L60 , msl1γ269 and msl11 ) and wild type control. The number of flies counted in this experiment was 2,100.

(b) Relative % viability of the female offspring with (dark brown bar) and without (light brown bar) maternally deposited MSL1. msl1L60 or msl1γ269/CyO, GFP flies were used as internal control with 100% viability. The mean ± SD of three independent biological replicates is presented. A total of 1435 flies were counted.

(c) Relative % viability of adult male (black bar) and female (brown bar) flies upon tubGal4-induced RNA silencing of msl1. UAS-msl1RNAi/TM6Tb flies not expressing dsRNA served as internal control with 100% viability. The mean ± SD of three biological replicates is shown. The number of flies counted in this experiment was 1,607.

Supplementary Figure 4 Genetic and biochemical characterization of MSL1-CDK7 interaction.

(a) Top panel: viability of flies expressing CDK7 from a temperature sensitive (ts) allele in a Cdk7 null mutant background (males) or heterozygous background (females) in the presence of wild type (CyOGFP/+) MSL1 levels (black bars) versus halved (msl1L60/+) MSL1 levels (yellow bars).

Bottom panel: comparison of the number of flies surviving to adulthood is shown for males and females ectopically expressing CDK7 from Cdk7ts in the absence of endogenous Cdk7 but wild type (CyOGFP/+) msl1 (black bars) and males ectopically expressing CDK7 from Cdk7ts in the presence of a mutation (If) that does not affect the dosage compensation process (yellow bars). The mean ± SD of three independent experiments is shown. The number of flies counted in this experiment was 730.

(b) Time course for CDK7 inhibitor (BS-181) treatment in D. melanogaster S2 cells. Pol II Ser5p levels are reduced after 5 or 15 min of treatment in comparison to DMSO control (lanes 1-6). MSL1, MOF and MSL3 are still able to co-IP after 15 min of BS-181 treatment (compare lanes 7 with 8 and 9) indicating that BS-181 does not disrupt interaction of MSL1 with the MSL proteins. No significant interaction is observed for MSL1 with Pol II RPB3 or Pol II Ser5p.

(c) Salivary glands dissected from wild type third instar larvae were treated with DMSO (negative control), or BS-181 (50μM) for 10min. Polytene chromosomal squashes were immunostained with Anti-rat-MSL1 (red) and Anti-Pol II (green). Asterisks indicate reduction in MSL1 signal in BS-181 treated samples compared to DMSO.

Supplementary Figure 5 Characterization of MSL1 phosphorylation in vivo.

(a) Annotated CID (collision induced dissociation) MS/MS spectrum of a tryptic peptide derived from the D. melanogaster MSL1 protein showing phosphorylation (pS) at amino acid (aa) S18. The peptide sequence is extensively covered by y and b fragment ions. Most of the fragment ions harboring the phosphoserine residue exhibit a neutral loss of phosphoric acid (-H3PO4), a commonly observed gas phase reaction pathway for CID fragmentation of phosphopeptides. The inset (right) of the figure illustrates the doubly charged (MH22+) peptide ion MS1 spectrum (Orbitrap-FTMS) measured with a very low mass deviation (MD).

(b) MSL1 is phosphorylated in vivo. S2 cells were cultured for an “in vivo labeling assay” in the presence of γATP32 and whole cell extracts were prepared. IP was performed with antibodies against N- and C- terminal domains of MSL1. Beads and pre-immune serum were used as negative controls. In vivo phosphorylated MSL1 was detected by autoradiography.

Supplementary Figure 6 Characterization of Anti-MSL1-PH1.

(a) Dot blot: different amounts of unmodified peptide, modified (phosphorylated) peptide at S18, MSL1-WT alone (-CAK), or with CAK and ATP (+CAK) were spotted on Protran BA79 nitrocellulose membrane (0.1μm). Detection was performed using Anti-MSL1-PH1.

(b) Whole cell extracts from S2 cells after RNAi against GFP or MSL1 were used for immunoprecipitation using Anti-MSL1 (lanes 5 and 6) and Anti-MSL1-PH1 (lanes 7 and 8). Agarose A beads (lanes 3 and 4) and pre-immune serum (lanes 9 and 10) were used as controls. Input shows 1% of whole cell extract. WB analysis using Anti-MSL1 shows that the signal detected in the MSL1-PH1 IP is decreased upon MSL1-RNAi (compare lanes 7 and 8) highlighting the specificity of Anti-MSL1-PH1. WB analysis using Anti-MOF showed that MSL1 phosphorylated at S18 interacts with MOF and this signal is also decreased upon MSL1-RNAi.

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Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Note (PDF 1632 kb)

Supplementary Data Set 1

Uncropped western blots and Coomassie-stained gels (PDF 856 kb)

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Chlamydas, S., Holz, H., Samata, M. et al. Functional interplay between MSL1 and CDK7 controls RNA polymerase II Ser5 phosphorylation. Nat Struct Mol Biol 23, 580–589 (2016). https://doi.org/10.1038/nsmb.3233

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