1. Volume 2, Issue 5, Pages 1068-1076 (November 2012)
Cdk11-CyclinL Controls the Assembly of the RNA Polymerase II Mediator Complex 
Julie Drogat, Valérie Migeot, Elise Mommaerts, Caroline Mullier, Marc Dieu, Harm van Bakel, Damien Hermand 
Cell Reports 
Volume 2, Issue 5, Pages (November 2012)
DOI: /j.celrep
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2. Cell Reports 2012 2, 1068-1076DOI: (10.1016/j.celrep.2012.09.027)
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3. Figure 1
A Cdk11-Cyclin L Complex Is Present in Fission Yeast and Affects Transcription Independently of CTD Phosphorylation
(A) Cdk11-GFP expressed from the endogenous locus or an untagged control were observed by fluorescence microscopy in the presence of DAPI.
(B) The cdk11-TAP strain was synchronized using a cdc25-22 block and release. At indicated times, samples were processed for TAP western blot and fluorescence-activated cell sorting analysis. The replicated population is marked in red.
(C) TAP of the cdk11-TAP and an untagged strain. Proteins identified from bands by mass spectrometry are indicated.
(D) Strains harboring the indicated tagged proteins were lysed and the Lcp1-TAP protein was precipitated on immunoglobulin G (IgG) beads. Total or precipitated products were resolved on PAGE and analyzed by western blot using the indicated antibodies. The asterisk indicates the IgG heavy chains.
(E) Strains harboring the indicated tagged proteins were lysed and immunoprecipitation was performed on IgG beads followed by kinase assay on GST-CTD. The kinase gel was stained with Coomassie blue. western blot analysis was performed using the peroxidase antiperoxidase antibody.
(F) The indicated strains were grown 2 hr in the presence or absence of the 3MB-PP1 inhibitor, lysed, and separated by PAGE. Western blot analyses were performed with the indicated antibodies.
See also Figure S2.
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4. Figure 2
The Cdk11 and Mediator-Associated Cdk8 Kinase Regulate a Common Set of Genes
(A) Left: Average ChIP-binding profiles of genes that were significantly bound (binding ratio ≥2, and p < 0.05) by Cdk11-HA (blue) and Rpb3-HA (red). The 95% confidence intervals and a graphical representation of the transcribed units (Gene) are displayed. Middle: Plots showing the correlation between binding ratios of ChIP-on-chips experiments performed with the indicated proteins (based on previous data (Coudreuse et al., 2010), Cdk7 is SpMcs6 and Cdk12 is SpLsk1). The correlation coefficients for each pair-wise combination are indicated in the bottom right corner of the scatter plots. Right: Occupancy intersection of ChIP-binding peaks for the Cdk11/Rpb3 combination (ALL) and their repartition between coding regions (CDS) and intergenic regions (IGR) with associated p values obtained from hypergeometric tests.
(B) ChIP-on-chip occupancy profiles of Rpb3-HA and Cdk11-HA along the SPBC947.04, SPAC , and SPCC genomic regions. Transcripts and open reading frames (ORFs) are indicated.
(C) Hierarchical clustering of the 90 mRNAs whose expression is significantly affected in the cdk8::ura4 or the cdk11::ura4 strains. Strains were grown in yeast extract supplemented (YES). The data are presented as a log2 mutant/wild-type ratio of hybridization signals and are color-coded, as indicated in the key (p value ≤ 0.05 and ≥1.5-fold change).
(D) Relative quantification (RQ) of the indicated mRNAs determined by quantitative RT-PCR using the ΔΔct method in wild-type, cdk8::ura4, cd11::ura4, and the double cdk8::ura4 cd11::ura4 mutants. a.u.: arbitrary units.
See also Figure S3.
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5. Figure 3
The Cdk11-Cyclin L Complex Phosphorylates Subunits of the Mediator Complex
(A) The Lcp1-TAP protein was precipitated and used on beads for in vitro kinase assay using various purified GST-fusion proteins as indicated. The amount of substrates was estimated by staining of the kinase gel.
(B) The Lcp1-TAP protein was precipitated and used on beads for in vitro kinase assay using various wild-type and mutated forms of the GST-Med4 protein as indicated. The conserved phosphorylation site (S115 in fission yeast) is shown on the right. The amount of substrates was estimated by staining of the kinase gel.
(C) The Lcp1-TAP protein was precipitated and used on beads for in vitro kinase assay using the wild-type and mutated forms of the GST-Med27 protein as indicated. The conserved phosphorylation site (S235 in fission yeast) is shown on the right. The amount of substrates was estimated by staining of the kinase gel. Note that this site is not conserved in budding yeast Med3, a putative ortholog of Med27.
See also Figure S4.
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6. Figure 4
Phosphorylation of Med4 and Med27 by Cdk11-Lcp1 Regulates the Association of the Kinase Module with the S-Mediator
(A) RQ of the indicated mRNAs determined by quantitative RT-PCR using the ΔΔct method in wild-type, med27 S235A (med27∗), med4 S115A S204A S218A (med4∗), and the double med27∗ med4∗ mutants. a.u.: arbitrary units.
(B) Strains harboring the indicated tagged proteins were lysed, and the TAP-tagged protein was precipitated on IgG beads. Total or precipitated products were resolved on PAGE and analyzed by western blot using the indicated antibodies. Note that both the Med27 S235A (Med27∗) and the Med4 S115A S204A S218A (Med4∗) mutants are myc-tagged. All relevant combinations were run on the same gel. Unprocessed blot images are presented in Figure S1A.
(C) The occupancy of Cdk8-TAP, Med13-TAP, and Med7-TAP was measured by ChIP at indicated loci in a wild-type strain and a cdk11::ura4 mutant. The error bars were calculated from duplicates.
(D) A model of the association of the kinase module with the S-Mediator regulated by Cdk11-Lcp1 phosphorylation of Med27 and Med4.
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7. Figure S1
Conservation of the Mediator During Evolution, Related to Figure 4
(A) Crude unprocessed ImageQuant (GE Healthcare) pictures of the western blots presented in Figure 4B. The strain number is indicated and refers to the strain list. Relevant information on the genotype is also indicated.
(B) Conservation of the Mediator subunits in S. cerevisiae, S. pombe, and human. The color code referring to the Mediator architecture is indicated.
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8. Figure S2
The Cdk11-Lcp1 Complex Is Related to Metazoan Cdk11-Cyclin L, Related to Figure 1
(A) Sequence alignment of the Cdk11 protein in various species. The PITSLRE motif is highlighted in red. The “gatekeeper” site mutated in the analog-sensitive kinase (M152G) is marked in green. ∗ indicates identity, : indicates high similarity, . indicates similarity. Hs, Homo sapiens; Mm, Mus musculus; Dr, Danio rerio; Dm, Drosophila melanogaster; Sp, Schizosaccharomyces pombe.
(B) Schematics of the domains found in the Cdk11 protein in various species, see A. RE refers to an arginine-glutamic acid rich region. The PITSLRE motif and the length of the sequences are indicated.
(C) Sequence alignment of the Lcp1 (cyclin L) protein in various species. ∗ indicates identity, : indicates high similarity, . indicates similarity. Hs, Homo sapiens; Mm, Mus musculus; Dr, Danio rerio; Dm, Drosophila melanogaster; Sp, Schizosaccharomyces pombe.
(D) Schematics of the domains found in the Lcp1 (cyclin L) protein in various species, see C. RS refers to an arginine-serine rich region. The cyclin box motif and the length of the sequences are indicated.
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9. Figure S3
Cdk11 Is Largely Distributed Onto Chromatin and Affects the Expression of a Subset of Genes, Related to Figure 2
(A) ChIP-on-chip occupancy profiles of Rpb3-HA and Cdk11-HA along the SPBPB2B2.06c, SPBPB7E8.01, SPAC25B8.13c, SPAC9E9.09c, SPAC186.01, SPAC343.12, and SPAC23D3.12 genomic regions. Transcripts and ORFs are indicated.
(B) Hierarchical clustering of 1896 mRNAs, whose expression is significantly affected in at least one of the lsk1::ura4, cdk11::ura4, cdk8::ura4, cdk9-as, or mcs6-as (cdk7) strains. Strains were grown in YES. The data are presented as log2 mutant/wild-type ratio of hybridization signals and are color-coded as indicated in the key of Figure S2C (p value ≤ 0.05 and ≥ 1.5-fold change).
(C) Hierarchical clustering of the 10 more strongly affected mRNAs in the cdk11::ura4 strain and their level in the cdk8::ura4 strain. Strains were grown in YES. The data are presented as log2 mutant/wild-type ratio of hybridization signals and are color-coded as indicated in the key (p value ≤ 0.05 and ≥ 1.5-fold change).
(D) Generation of an -as mutant of Cdk11. The -as mutation is M152G. Strains expressing Cdk11-TAP (wt or –as, as indicated) were lysed, and immunoprecipitation was performed on IgG beads followed by kinase assay on Histone H1 (Calbiochem) or Myelin Basic Protein (MBP, Calbiochem) in the presence of labeled ATP of labeled N6-benzyl-ATP (N6B), as indicated. As control, Cdc2 was precipitated using a monoclonal antibody (a kind gift of Paul Nurse). Note that Cdk11 autophosphorylates in the presence of labeled ATP, but not N6B.
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10. Figure S4
The Cdk11-Cyclin L Complex Phosphorylates Subunits of the Mediator Complex, Related to Figure 3
(A) The integrated fission yeast ORFeome is a collection of strains, each expressing a different Flag-tagged ORF from the leu1 locus. We recovered the 23 flag-tagged Mediator subunits containing strains from the library. Each of the flag-tagged proteins and the Lcp1-TAP protein (or an untagged strain as indicated) were precipitated, mixed, and used on beads for in vitro kinase assay. Only a subset of the subunits (med14-flag, med27-flag, med4-flag, med7-flag, med1-flag, and med19-flag) are shown. The coprecipitated Cdk11 autophosphorylates (marked by °) and bands specifically phosphorylated when both a flag-tagged protein and Cdk11-Lcp1 were present are indicated by an asterisk. For each flag-tagged Mediator subunit, anti-flag western blots confirmed the presence of the protein (data not shown).
(B) Sequence alignment of the Med4 protein in various species. The S115, S204, and S218 residues are highlighted in red. ∗ indicates identity, : indicates high similarity, . indicates similarity. Hs, Homo sapiens; Mm, Mus musculus; Dr, Danio rerio; Dm, Drosophila melanogaster; Sp, Schizosaccharomyces pombe.
(C) Sequence alignment of the Med27 protein in various species and the proposed budding yeast ortholog Med3. The S235 residue is highlighted in red. ∗ indicates identity, : indicates high similarity, . indicates similarity. Hs, Homo sapiens; Mm, Mus musculus; Dr, Danio rerio; Dm, Drosophila melanogaster; Sp, Schizosaccharomyces pombe.
Cell Reports 2012 2, DOI: ( /j.celrep )
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11. Figure S5
Error in the cdk8 Tagging Primers on Pombe PCR Primer Program
While this work was in progress, we noticed a mistake in the automatic primer design software (PPPP, Pombe PCR Primer Program [ The software does not take into account the presence of an intron at the end of the cdk8 sequence, which generates a truncated tagged protein that is poorly expressed.
(A) Wrong Cdk8 protein sequence with the unspliced intron indicated in lowercase.
(B) Correct Cdk8 protein sequence.
Cell Reports 2012 2, DOI: ( /j.celrep )
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