Protein interactions within the Set1 complex and their roles in the regulation of histone 3 lysine 4 methylation

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5′ end of active coding regions but a decrease of H3K4 dimethylation at the 3′ end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3′ end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.Acciones Integradas Hispano-Francesas HF2003-0170Ministerio de Educación y Ciencia BFU2005-02603Ministerio de Ciencia y Tecnología BMC2003- 07072-C03-0

Regulation of Mus81-Eme1 structure-specific endonuclease by Eme1 SUMO-binding and Rad3ATR kinase is essential in the absence of Rqh1BLM helicase

International audienceThe Mus81-Eme1 structure-specific endonuclease is crucial for the processing of DNA recombination and late replication intermediates. In fission yeast, stimulation of Mus81-Eme1 in response to DNA damage at the G2/M transition relies on Cdc2 CDK1 and DNA damage checkpoint-dependent phosphorylation of Eme1 and is critical for chromosome stability in absence of the Rqh1 BLM helicase. Here we identify Rad3 ATR checkpoint kinase consensus phosphorylation sites and two SUMO interacting motifs (SIM) within a short N-terminal domain of Eme1 that is required for cell survival in absence of Rqh1 BLM. We show that direct phosphorylation of Eme1 by Rad3 ATR is essential for catalytic stimulation of Mus81-Eme1. Chk1-mediated phosphorylation also contributes to the stimulation of Mus81-Eme1 when combined with phosphorylation of Eme1 by Rad3 ATR. Both Rad3 ATR-and Chk1-mediated phosphorylation of Eme1 as well as the SIMs are critical for cell fitness in absence of Rqh1 BLM and abrogating bimodal phosphorylation of Eme1 along with mutating the SIMs is incompatible with rqh1Δ cell viability. Our findings unravel an elaborate regulatory network that relies on the poorly structured N-terminal domain of Eme1 and which is essential for the vital functions Mus81-Eme1 fulfills in absence of Rqh1 BLM

Regulation of Mus81–Eme1 Holliday junction resolvase in response to DNA damage

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Structural Characterization of Set1 RNA Recognition Motifs and their Role in Histone H3 Lysine 4 Methylation

International audienceThe yeast Set1 histone H3 lysine 4 (H3K4) methyltransferase contains, in addition to its catalytic SET domain, a conserved RNA recognition motif (RRM1). We present here the crystal structure and the secondary structure assignment in solution of the Set1 RRM1. Although RRM1 has the expected betaalphabetabetaalphabeta RRM-fold, it lacks the typical RNA-binding features of these modules. RRM1 is not able to bind RNA by itself in vitro, but a construct combining RRM1 with a newly identified downstream RRM2 specifically binds RNA. In vivo, H3K4 methylation is not affected by a point mutation in RRM2 that preserves Set1 stability but affects RNA binding in vitro. In contrast mutating RRM1 destabilizes Set1 and leads to an increase of dimethylation of H3K4 at the 5'-coding region of active genes at the expense of trimethylation, whereas both, dimethylation decreases at the 3'-coding region. Taken together, our results suggest that Set1 RRMs bind RNA, but Set1 RNA-binding activity is not linked to H3K4 methylation

Cotranslational assembly of the yeast SET1C histone methyltransferase complex

While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein–RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation

Control of structure-specific endonucleases to maintain genome stability

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