Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Apr 21;62(2):207-221.
doi: 10.1016/j.molcel.2016.03.016.

SHREC Silences Heterochromatin via Distinct Remodeling and Deacetylation Modules

Godwin Job  1 , Christiane Brugger  2 , Tao Xu  1 , Brandon R Lowe  1 , Yvan Pfister  2 , Chunxu Qu  3 , Sreenath Shanker  1 , José I Baños Sanz  2 , Janet F Partridge  4 , Thomas Schalch  5
Affiliations

SHREC Silences Heterochromatin via Distinct Remodeling and Deacetylation Modules

Godwin Job et al. Mol Cell. .

Abstract

Nucleosome remodeling and deacetylation (NuRD) complexes are co-transcriptional regulators implicated in differentiation, development, and diseases. Methyl-CpG binding domain (MBD) proteins play an essential role in recruitment of NuRD complexes to their target sites in chromatin. The related SHREC complex in fission yeast drives transcriptional gene silencing in heterochromatin through cooperation with HP1 proteins. How remodeler and histone deacetylase (HDAC) cooperate within NuRD complexes remains unresolved. We determined that in SHREC the two modules occupy distant sites on the scaffold protein Clr1 and that repressive activity of SHREC can be modulated by the expression level of the HDAC-associated Clr1 domain alone. Moreover, the crystal structure of Clr2 reveals an MBD-like domain mediating recruitment of the HDAC module to heterochromatin. Thus, SHREC bi-functionality is organized in two separate modules with separate recruitment mechanisms, which work together to elicit transcriptional silencing at heterochromatic loci.

PubMed Disclaimer

Figures

Figure 1
Figure 1. SHREC subunits show separation of function in gene expression
(A) Scheme of SHREC complex. (B–E) RNA-seq analysis of strains deleted for individual SHREC components. (B) Representation of number of reads from repetitive sequences located at the centromeres in different genetic backgrounds normalized to WT cells. (C) Unsupervised Hierarchical Clustering Analysis of the top 1000 most variable genes based on MAD score (median absolute deviation). Blue lines represent individual transcripts whose expression is low, and red those that are highly expressed. (D) Venn diagram showing overlap in differentially regulated genes (>1.5 fold difference, <0.05 FDR). Blue numbers reflect overlap between 2 genotypes, red between 3 genotypes, white between 4 genotypes and black between 5 genotypes. (E) Representative genes whose transcripts are differentially regulated between clr1Δ, clr2Δ and clr3Δ compared with mit1Δ and chp2Δ (>1.5 fold change and FDR <0.05). Q-RT-PCR (left panel) confirms RNA-seq patterns (right panel). Red are sense and blue are antisense transcript ratios shown as log2 ratio, with a scale of −2 to +2. Signal above the line represents overexpression, and below is repression relative to WT. See also Fig. S1.
Figure 2
Figure 2. The C-terminal end of Clr1 is the assembly platform for the SHREC HDAC module
(A) Serial dilution growth assay of clr1Δ cells expressing various domains of Clr1 (grey bars). Strains were assessed for growth on PMG media lacking leucine (PMG-leu) to select for maintenance of the plasmid, PMG-leu-ura to monitor cen∷ura4+ expression and PMG-leu+FOA to monitor silencing of cen1∷ura4+. The overexpressed Clr1T fragment is required and sufficient for silencing at cen1∷ura4+. (B–C) Serial dilution assay of strains bearing genomic clr1 truncation mutants assessed for silencing of (B) cen1∷ura4+ and (C) mat3M∷ura4+.clr1 truncations expressed at endogenous levels do not confer proper silencing at either locus. (D–G) Immunoprecipitation and western blot analyses reveal (D) Clr2 interacts strongly with Clr1T even in the absence of Clr3. (E) Clr1T binding to Clr3 is dependent on the presence of Clr2. (F) Clr3 binding to Clr2 is dependent on the presence of Clr1. (G) Clr3 binding appears to depend on both Zn finger and C terminal domains of Clr1T. See also Fig. S2.
Figure 3
Figure 3. Mit1 interacts with Clr1 350–500 using C-terminal zinc finger motifs
(A) Schematic view of domains forming the interaction between Mit1 and Clr1. Residues observed in the crystal structure as Mit1 interaction domain (MID) on Clr1 and Clr1 interaction domain (CID) on Mit1 are shown as gray bars. The two zinc fingers (ZnF) identified as part of the Mit1 CID are indicated. (B) Surface representation of the Mit1-Clr1 interface shows the two proteins Mit1 (orange) and Clr1 (green) twisting around each other. (C) Basic amino acids covering the surface of the Clr1-Mit1 complex are shown in stick representation (orientation as in (B), right side). Zoom outs highlight the two zinc fingers of the Mit1 fold. (D) Electrostatic surface representation of Clr1-Mit1 complex in same orientation as in (C). (E) EMSA assay of radiolabelled cen75 DNA analyzed on 5% polyacrylamide gel. (F) Immunoprecipitation and western blot analyses reveal Mit1 binds to full length but not to Clr1T. See also Fig. S3.
Figure 4
Figure 4. Crystal structure of the Clr1-Clr2 complex shows Clr1 wrapping around the multidomain protein Clr2
(A) Surface representation of Clr2 in white with Clr1 in cartoon representation shown in green. Dashed lines indicate loops not observed in the electron density. (B) Clr1-Clr2 structure in cartoon representation colored by domain assignment as defined in the linear schematic (left). Zoom out shows structural zinc motif inside the Clr2 NTD. (C) Serial dilution growth assays of clr2Δ cells expressing Clr2 truncation constructs (indicated schematically by grey bars) show that all Clr2 domains are required for function. (D) IP of Clr2 constructs in (C) by Clr1T and probing for interaction of Clr2 and Clr3 by Western blot. (E) Serial dilution assays of Clr1T mutants (indicated schematically) expressed in clr1Δ cells show that α1 and β1 domains are required for function. (F) IP of Clr1T constructs in (E) and probing for interaction with Clr2 and Clr3 by Western blot. See also Fig. S4.
Figure 5
Figure 5. Clr1-2 contains a MBD-like domain and binds to dsDNA and RNA
(A) MBD4 structure with DNA (PDB ID: 3VYQ, orange) superimposes onto Clr2 N-terminal domain with RMDS of 2.8 Å. (B) EMS A of the wild type and the MBD6A mutant Clr1-Clr2 complex on 5% acrylamide gel. Nucleic acid substrates are 5'-P32 labeled cen75 dsDNA or dsRNA. (C) Competition of 32P labeled cen75 dsRNA with non-labeled dsRNA and DNA and single stranded RNA and DNA. (D) Serial dilution growth assays of Clr2 MBDL mutants show that the MBDL6A mutant loses silencing function. (E) IP and Western blot analysis shows that the Clr2MBDL6A mutant maintains interaction with Clr1T and Clr3. (F) ChIP for Clr2 at centromeric dh repeat shows that Clr2 is recruited by MBDL domain. See also Fig. S5.
Figure 6
Figure 6. Clr3 HDAC dimer uses the α/β-hydrolase-based Arb2 domain for silencing through SHREC
(A) Clr3 structure with HDAC domains on top and Arb2 domains at the bottom. One of the Clr3 monomers (the asymmetric unit) is colored blue (HDAC) and yellow (Arb2 domain). Zinc ions are shown as gray, potassium as purple and magnesium as green spheres. (B) Schematic of Clr3-3xFLAG tagged mutants. (C) Comparative growth assay of serially diluted clr3Δ strains bearing a cen∷ura4+ reporter, which have been transformed with Clr3-3xFLAG tagged mutants. Strains were assessed for growth on PMG media lacking histidine (PMG-HIS) to select for maintenance of the plasmid, PMG-HIS-URA to monitor cen∷ura4+ expression and PMG-HIS+FOA to monitor silencing of cen∷ura4+. Truncation mutants (left panel) and structure-based mutants (right panel) were assayed. Integrity of Arb2 domain is required for silencing. (D) IP of Clr1 and analysis by Western blot identifies Arb2 mutants losing interaction with Clr1. See also Fig. S6.
Figure 7
Figure 7. SHREC is a flexibly linked assembly of functional modules
Model of the SHREC complex assembled from the crystal structures presented in this work and the structure of the chromo and helicase domains of Chd1 (Hauk et al. 2010). SHREC structurally and functionally divides into a remodeling module and an HDAC module linked by Clr1. While the remodeling module is recruited through Chp2 the HDAC module is recruited through the nucleic acid binding activity of the MBDL of Clr2.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Allen HF, Wade PA, Kutateladze TG. The NuRD architecture. Cell. Mol. Life Sci. 2013;70:3513–3524. - PMC - PubMed
    1. Allshire RC, Nimmo ER, Ekwall K, Javerzat JP, Cranston G. Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation. Genes Dev. 1995;9:218–233. - PubMed
    1. Alper BJ, Lowe BR, Partridge JF. Centromeric heterochromatin assembly in fission yeast--balancing transcription, RNA interference and chromatin modification. Chromosome Res. 2012;20:521–534. - PMC - PubMed
    1. Alper BJ, Job G, Yadav RK, Shanker S, Lowe BR, Partridge JF. Sir2 is required for Clr4 to initiate centromeric heterochromatin assembly in fission yeast. EMBO J. 2013;32:2321–2335. - PMC - PubMed
    1. Aygün O, Mehta S, Grewal SIS. HDAC-mediated suppression of histone turnover promotes epigenetic stability of heterochromatin. Nat. Struct. Mol. Biol. 2013;20:547–554. - PMC - PubMed

MeSH terms