Identification of Functional Domains in the Cohesin Loader Subunit Scc4 by a Random Insertion/Dominant Negative Screen

Cohesin is a multi-subunit complex that plays an essential role in genome stability. Initial association of cohesin with chromosomes requires the loader—a heterodimer composed of Scc4 and Scc2. However, very little is known about the loader’s mechanism of action. In this study, we performed a genetic screen to identify functional domains in the Scc4 subunit of the loader. We isolated scc4 mutant alleles that, when overexpressed, have a dominant negative effect on cell viability. We defined a small region in the N terminus of Scc4 that is dominant negative when overexpressed, and on which Scc2/Scc4 activity depends. When the mutant alleles are expressed as a single copy, they are recessive and do not support cell viability, cohesion, cohesin loading or Scc4 chromatin binding. In addition, we show that the mutants investigated reduce, but do not eliminate, the interaction of Scc4 with either Scc2 or cohesin. However, we show that Scc4 cannot bind cohesin in the absence of Scc2. Our results provide new insight into the roles of Scc4 in cohesin loading, and contribute to deciphering the loading mechanism

Gene Silencing by RNA Interference in the White Rot Fungus Phanerochaete chrysosporium▿

The effectiveness of RNA interference (RNAi) is demonstrated in the lignin-degrading fungus Phanerochaete chrysosporium. The manganese-containing superoxide dismutase gene (MnSOD1) was used as the target for RNAi. The plasmid constructed for gene silencing contained a transcriptional unit for hairpin RNA expression. Significantly lower MnSOD expression at both the mRNA and protein activity levels was detected in RNAi transformants. Furthermore, even though P. chrysosporium possesses three copies of the MnSOD gene, this RNAi construct was sufficient to decrease the enzymatic activity by as much as 70% relative to control levels. Implementation of the RNAi technique in P. chrysosporium provides an alternative genetic tool for studies of gene function, particularly of essential genes or gene families

A Conserved Domain in the Scc3 Subunit of Cohesin Mediates the Interaction with Both Mcd1 and the Cohesin Loader Complex

<div><p>The Structural Maintenance of Chromosome (SMC) complex, termed cohesin, is essential for sister chromatid cohesion. Cohesin is also important for chromosome condensation, DNA repair, and gene expression. Cohesin is comprised of Scc3, Mcd1, Smc1, and Smc3. Scc3 also binds Pds5 and Wpl1, cohesin-associated proteins that regulate cohesin function, and to the Scc2/4 cohesin loader. We mutagenized <i>SCC3</i> to elucidate its role in cohesin function. A 5 amino acid insertion after Scc3 residue I358, or a missense mutation of residue D373 in the adjacent stromalin conservative domain (SCD) induce inviability and defects in both cohesion and cohesin binding to chromosomes. The I358 and D373 mutants abrogate Scc3 binding to Mcd1. These results define an Scc3 region extending from I358 through the SCD required for binding Mcd1, cohesin localization to chromosomes and cohesion. Scc3 binding to the cohesin loader, Pds5 and Wpl1 are unaffected in I358 mutant and the loader still binds the cohesin core trimer (Mcd1, Smc1 and Smc3). Thus, Scc3 plays a critical role in cohesin binding to chromosomes and cohesion at a step distinct from loader binding to the cohesin trimer. We show that residues Y371 and K372 within the SCD are critical for viability and chromosome condensation but dispensable for cohesion. However, scc3 Y371A and scc3 K372A bind normally to Mcd1. These alleles also provide evidence that Scc3 has distinct mechanisms of cohesin loading to different loci. The cohesion-competence, condensation-incompetence of Y371 and K372 mutants suggests that cohesin has at least one activity required specifically for condensation.</p></div

Developing a peptide to disrupt cohesin head domain interactions

Summary: Cohesin mediates the 3-D structure of chromatin and is involved in maintaining genome stability and function. The cohesin core comprises Smc1 and Smc3, elongated-shaped proteins that dimerize through globular domains at their edges, called head and hinge. ATP binding to the Smc heads induces their dimerization and the formation of two active sites, while ATP hydrolysis results in head disengagement. This ATPase cycle is essential for driving cohesin activity. We report on the development of the first cohesin-inhibiting peptide (CIP). The CIP binds Smc3 in vitro and inhibits the ATPase activity of the holocomplex. Treating yeast cells with the CIP prevents cohesin’s tethering activity and, interestingly, leads to the accumulation of cohesin on chromatin. CIP3 also affects cohesin activity in human cells. Altogether, we demonstrate the power of peptides to inhibit cohesin in cells and discuss the potential application of CIPs as a therapeutic approach

Scc3 does not affect the Smc3-Mcd1 interaction.

<p><b>A.</b> Strain YOG3049 (<i>SCC3-AID-V5 SMC3-6HA</i>) was grown to the mid-log phase. Cells were divided into two flasks and 1 mM IAA was added to one of them for 60 minutes. The depletion of Scc3-3V5 was measured in the extract by Western blot with antibodies against V5. Un-depleted and depleted Scc3 cells were used for immunoprecipitation of Smc3-6HA with anti-HA antibodies. The co-precipitation of Mcd1 was detected by anti-Mcd1 antibody. <b>B</b>. Strain YOG3027 (<i>SCC3-AID-V5 SCC2-12MYC</i>) was grown to the mid-log phase. Cells were divided into two flasks and 1 mM IAA was added to one of them for 120 minutes. The depletion of Scc3-3V5 was measured in the extract by Western blot with antibodies against V5. Cells were used for immunoprecipitation of Scc2-12Myc with anti-MYC antibodies. The co-precipitation of Mcd1 was detected by anti-Mcd1 antibody.</p

Analysis of chromosome condensation in Scc3 point mutants.

<p><b>A.</b> Strains YOG3021 (<i>SCC3-6HA scc3-6</i>), YOG3024 (<i>scc3-Y371A-6HA scc3-6</i>) and YOG3025 (<i>scc3-K372A-6HA scc3-6</i>), were grown at permissive temperature (23°C) and arrested at G1 using α-factor. The cells were shifted to restrictive temperature (35.5°C) and re-arrested in the G2/M phase using nocodazole. Nuclei were spread and the bulk DNA was stained with DAPI. The morphology of the <i>rDNA</i>-loop of 100 nuclei of each strain was scored (n = 3). <b>B.</b> Representative photomicrographs of wild-type <i>rDNA</i> loops (left panel) and condensation defects at the <i>rDNA</i> locus. Arrow indicates the rDNA region.</p

scc3 Y371 binding to the <i>rDNA</i> is reduced.

<p>Strains YOG3021 (<i>SCC3-6HA scc3-6</i>) and YOG3024 (<i>scc3-Y371A-6HA scc3-6</i>) were processed for chromatin immunoprecipitation analysis. Scc3 was immunoprecipitated with anti-HA antibodies and the precipitated DNA was analyzed by quantitative PCR for: <b>A.</b> chromosome III CARC1; <b>B.</b> chromosome IV centromere; <b>C.</b> chromosoXII <i>rDNA</i>. A representative PCR is shown (n = 3).</p

Co-immunoprecipitation of Scc3-I358Ins with core and accessory cohesin subunits.

<p><b>A.</b> Analysis of scc3-I358ins-6HA binding to Mcd1, Scc2 and Pds5. Haploids JH5257 (<i>SCC2-3V5</i>), YIO92 (<i>SCC2-3V5 SCC3-6HA</i>) or YIO92R1 (<i>SCC2-3V5 scc3-I358ins-6HA</i>) cells were grown to mid-log phase in YPD media, lysed and subjected to immunoprecipitation against the HA tag (Scc3). Precipitated proteins were analyzed by Western blot using antibodies against HA (IP), Mcd1 (co-IP), V5 (co-IP) and Pds5 (co-IP). <b>B.</b> Analysis of scc3-I358ins-6HA binding to Wpl1. VG3333 (<i>WPL1-3V5</i>) YOG3007 (<i>WPL1-3V5 SCC3-6HA</i>) or YOG3008 (<i>WPL1-3V5 scc3-I358ins-6HA</i>) cells were analyzed as described in A. Precipitated proteins were analyzed by Western blot using antibodies against HA (IP) and V5 (co-IP).</p

Identification of a functional domain in Scc3.

<p><b>A.</b> Schematic of the cohesin complex. The two yellow balls between the Smc1 and Smc3 represent ATP molecules. <b>B.</b> Flowchart of the experimental design to identify <i>SCC3</i> dominant negative mutants. <b>C.</b> Strain VG3135 (<i>scc3-6</i>) cells carrying pRS406 (<i>pGALURA3</i>), pIO88 (<i>pGAL</i>-<i>SCC3 URA3</i>) or pIO88R1 (<i>pGAL</i>-<i>scc3-I358insURA3</i>) were grown to saturation in SC-URA media. Fivefold serial dilutions of each strain was plated on SC-URA plates containing either glycerol or galactose and grown at 23°C. <b>D.</b> Sequence alignment of Scc3 was generated by ClustalX. The arrow indicates the insertion site after I358. The blue and red frames indicate the RID A region G350-I359 and the conserved sequence F367-D373, respectively.</p