Département de biologie moléculaire
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Schalch group

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Capturing Structural Heterogeneity in Chromatin Fibers.
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J Mol Biol, ; 429 (20): 3031-3042
Chromatin fiber organization is implicated in processes such as transcription, DNA repair and chromosome segregation, but how nucleosomes interact to form higher-order structure remains poorly understood. We solved two crystal structures of tetranucleosomes with approximately 11-bp DNA linker length at 5.8 and 6.7 Å resolution. Minimal intramolecular nucleosome-nucleosome interactions result in a fiber model resembling a flat ribbon that is compatible with a two-start helical architecture, and that exposes histone and DNA surfaces to the environment. The differences in the two structures combined with electron microscopy reveal heterogeneous structural states, and we used site-specific chemical crosslinking to assess the diversity of nucleosome-nucleosome interactions through identification of structure-sensitive crosslink sites that provide a means to characterize fibers in solution. The chromatin fiber architectures observed here provide a basis for understanding heterogeneous chromatin higher-order structures as they occur in a genomic context.


SHREC Silences Heterochromatin via Distinct Remodeling and Deacetylation Modules.
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Mol Cell, ; 62 (2): 207-221
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.


Panspecies small-molecule disruptors of heterochromatin-mediated transcriptional gene silencing.
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Mol Cell Biol, ; 35 (4): 662-674
Heterochromatin underpins gene repression, genome integrity, and chromosome segregation. In the fission yeast Schizosaccharomyces pombe, conserved protein complexes effect heterochromatin formation via RNA interference-mediated recruitment of a histone H3 lysine 9 methyltransferase to cognate chromatin regions. To identify small molecules that inhibit heterochromatin formation, we performed an in vivo screen for loss of silencing of a dominant selectable kanMX reporter gene embedded within fission yeast centromeric heterochromatin. Two structurally unrelated compounds, HMS-I1 and HMS-I2, alleviated kanMX silencing and decreased repressive H3K9 methylation levels at the transgene. The decrease in methylation caused by HMS-I1 and HMS-I2 was observed at all loci regulated by histone methylation, including centromeric repeats, telomeric regions, and the mating-type locus, consistent with inhibition of the histone deacetylases (HDACs) Clr3 and/or Sir2. Chemical-genetic epistasis and expression profiles revealed that both compounds affect the activity of the Clr3-containing Snf2/HDAC repressor complex (SHREC). In vitro HDAC assays revealed that HMS-I1 and HMS-I2 inhibit Clr3 HDAC activity. HMS-I1 also alleviated transgene reporter silencing by heterochromatin in Arabidopsis and a mouse cell line, suggesting a conserved mechanism of action. HMS-I1 and HMS-I2 bear no resemblance to known inhibitors of chromatin-based activities and thus represent novel chemical probes for heterochromatin formation and function.


CRL4-like Clr4 complex in Schizosaccharomyces pombe depends on an exposed surface of Dos1 for heterochromatin silencing.
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Proc Natl Acad Sci U S A, ; 111 (5): 1795-1800
Repressive histone H3 lysine 9 methylation (H3K9me) and its recognition by HP1 proteins are necessary for pericentromeric heterochromatin formation. In Schizosaccharomyces pombe, H3K9me deposition depends on the RNAi pathway. Cryptic loci regulator 4 (Clr4), the only known H3K9 methyltransferase in this organism, is a subunit of the Clr4 methyltransferase complex (CLRC), whose composition is reminiscent of a CRL4 type cullin-RING ubiquitin ligase (CRL) including its cullin Cul4, the RING-box protein Pip1, the DNA damage binding protein 1 homolog Rik1, and the DCAF-like protein delocalization of Swi6 1 (Dos1). Dos2 and Stc1 have been proposed to be part of the complex but do not bear similarity to canonical ubiquitin ligase components. CLRC is an active E3 ligase in vitro, and this activity is necessary for heterochromatin assembly in vivo. The similarity between CLRC and the CRLs suggests that the WD repeat protein Dos1 will act to mediate target recognition and substrate specificity for CLRC. Here, we present a pairwise interaction screen that confirms a CRL4-like subunit arrangement and further identifies Dos2 as a central component of the complex and recruiter of Stc1. We determined the crystal structure of the Dos1 WD repeat domain, revealing an eight-bladed β-propeller fold. Functional mapping of the putative target-binding surface of Dos1 identifies key residues required for heterochromatic silencing, consistent with Dos1's role as the specificity factor for the E3 ubiquitin ligase.


The Chp1-Tas3 core is a multifunctional platform critical for gene silencing by RITS.
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Nat Struct Mol Biol, ; 18 (12): 1351-1357
RNA interference (RNAi) is critical for the assembly of heterochromatin at Schizosaccharomyces pombe centromeres. Central to this process is the RNA-induced initiation of transcriptional gene silencing (RITS) complex, which physically anchors small noncoding RNAs to chromatin. RITS includes Ago1, the chromodomain protein Chp1, and Tas3, which forms a bridge between Chp1 and Ago1. Chp1 is a large protein with no recognizable domains, apart from its chromodomain. Here we describe how the structured C-terminal half of Chp1 binds the Tas3 N-terminal domain, revealing the tight association of Chp1 and Tas3. The structure also shows a PIN domain at the C-terminal tip of Chp1 that controls subtelomeric transcripts through a post-transcriptional mechanism. We suggest that the Chp1-Tas3 complex provides a solid and versatile platform to recruit both RNAi-dependent and RNAi-independent gene-silencing pathways for locus-specific regulation of heterochromatin.


High-affinity binding of Chp1 chromodomain to K9 methylated histone H3 is required to establish centromeric heterochromatin.
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Mol Cell, ; 34 (1): 36-46
In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.


The molecular architecture of the metalloprotease FtsH.
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Proc Natl Acad Sci U S A, ; 103 (9): 3066-3071
The ATP-dependent integral membrane protease FtsH is universally conserved in bacteria. Orthologs exist in chloroplasts and mitochondria, where in humans the loss of a close FtsH-homolog causes a form of spastic paraplegia. FtsH plays a crucial role in quality control by degrading unneeded or damaged membrane proteins, but it also targets soluble signaling factors like sigma(32) and lambda-CII. We report here the crystal structure of a soluble FtsH construct that is functional in caseinolytic and ATPase assays. The molecular architecture of this hexameric molecule consists of two rings where the protease domains possess an all-helical fold and form a flat hexagon that is covered by a toroid built by the AAA domains. The active site of the protease classifies FtsH as an Asp-zincin, contrary to a previous report. The different symmetries of protease and AAA rings suggest a possible translocation mechanism of the target polypeptide chain into the interior of the molecule where the proteolytic sites are located.


X-ray structure of a tetranucleosome and its implications for the chromatin fibre.
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Nature, ; 436 (7047): 138-141
DNA in eukaryotic chromosomes is organized in arrays of nucleosomes compacted into chromatin fibres. This higher-order structure of nucleosomes is the substrate for DNA replication, recombination, transcription and repair. Although the structure of the nucleosome core is known at near-atomic resolution, even the most fundamental information about the organization of nucleosomes in the fibre is controversial. Here we report the crystal structure of an oligonucleosome (a compact tetranucleosome) at 9 A resolution, solved by molecular replacement using the nucleosome core structure. The structure shows that linker DNA zigzags back and forth between two stacks of nucleosome cores, which form a truncated two-start helix, and does not follow a path compatible with a one-start solenoidal helix. The length of linker DNA is most probably buffered by stretching of the DNA contained in the nucleosome cores. We have built continuous fibre models by successively stacking tetranucleosomes one on another. The resulting models are nearly fully compacted and most closely resemble the previously described crossed-linker model. They suggest that the interfaces between nucleosomes along a single helix start are polymorphic.


Nucleosome arrays reveal the two-start organization of the chromatin fiber.
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Science, ; 306 (5701): 1571-1573
Chromatin folding determines the accessibility of DNA constituting eukaryotic genomes and consequently is profoundly important in the mechanisms of nuclear processes such as gene regulation. Nucleosome arrays compact to form a 30-nanometer chromatin fiber of hitherto disputed structure. Two competing classes of models have been proposed in which nucleosomes are either arranged linearly in a one-start higher order helix or zigzag back and forth in a two-start helix. We analyzed compacted nucleosome arrays stabilized by introduction of disulfide cross-links and show that the chromatin fiber comprises two stacks of nucleosomes in accord with the two-start model.


Chromatin fiber folding: requirement for the histone H4 N-terminal tail.
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J Mol Biol, ; 327 (1): 85-96
We have developed a self-assembly system for nucleosome arrays in which recombinant, post-translationally unmodified histone proteins are combined with DNA of defined-sequence to form chromatin higher-order structure. The nucleosome arrays obtained are highly homogeneous and sediment at 53S when maximally folded in 1mM or 100mM MgCl(2). The folding properties are comparable to established systems. Analytical ultracentrifugation is used to determine the consequence of individual histone tail domain deletions on array folding. Fully compacted chromatin fibers are obtained with any one of the histone tails deleted with the exception of the H4 N terminus. The region of the H4 tail, which mediates compaction, resides in the stretch of amino acids 14-19.