Structural insights into p300 regulation and acetylation-dependent genome organisation.

Histone modifications are deposited by chromatin modifying enzymes and read out by proteins that recognize the modified state. BRD4-NUT is an oncogenic fusion protein of the acetyl lysine reader BRD4 that binds to the acetylase p300 and enables formation of long-range intra- and interchromosomal interactions. We here examine how acetylation reading and writing enable formation of such interactions.

Differential phosphorylation of Clr4 by Cdk1 accompanies a histone H3 methylation switch that is essential for gametogenesis.

Methylation of histone H3 at lysine 9 (H3K9) is a hallmark of heterochromatin that plays crucial roles in gene silencing, genome stability, and chromosome segregation. In Schizosaccharomyces pombe, Clr4 mediates both di- and tri-methylation of H3K9. Although H3K9 methylation has been intensely studied in mitotic cells, its role during sexual differentiation remains unclear.

SUV39 SET domains mediate crosstalk of heterochromatic histone marks.

The SUV39 class of methyltransferase enzymes deposits histone H3 lysine 9 di- and trimethylation (H3K9me2/3), the hallmark of constitutive heterochromatin. How these enzymes are regulated to mark specific genomic regions as heterochromatic is poorly understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast and recent evidence suggests that ubiquitination of lysine 14 on histone H3 (H3K14ub) plays a key role in H3K9 methylation.

Breakers and amplifiers in chromatin circuitry: acetylation and ubiquitination control the heterochromatin machinery.

Eukaryotic genomes are segregated into active euchromatic and repressed heterochromatic compartments. Gene regulatory networks, chromosomal structures, and genome integrity rely on the timely and locus-specific establishment of active and silent states to protect the genome and provide the basis for cell division and specification of cellular identity. Here, we focus on the mechanisms and molecular machinery that establish heterochromatin in Schizosaccharomyces pombe and compare it with Saccharomyces cerevisiae and the mammalian polycomb system.

Chromatin fiber structural motifs as regulatory hubs of genome function?

Nucleosomes cover eukaryotic genomes like beads on a string and play a central role in regulating genome function. Isolated strings of nucleosomes have the potential to compact and form higher order chromatin structures, such as the well-characterized 30-nm fiber. However, despite tremendous advances in observing chromatin fibers it has not been possible to confirm that regularly ordered fibers represent a prevalent structural level in the folding of chromosomes.

Transcriptional gene silencing requires dedicated interaction between HP1 protein Chp2 and chromatin remodeler Mit1.

Heterochromatin protein 1 (HP1) proteins are key factors of eukaryotic heterochromatin that coordinate chromatin compaction and transcriptional gene silencing. Through their multivalency they act as adaptors between histone H3 Lys9 di/trimethyl marks in chromatin and effector complexes that bind to the HP1 chromoshadow domain. Most organisms encode for multiple HP1 isoforms and the molecular mechanisms that underpin their diverse functions in genome regulation remain poorly understood.

Structure of the replication regulator Sap1 reveals functionally important interfaces.

The mechanism by which specific protein-DNA complexes induce programmed replication fork stalling in the eukaryotic genome remains poorly understood. In order to shed light on this process we carried out structural investigations on the essential fission yeast protein Sap1. Sap1 was identified as a protein involved in mating-type switching in Schizosaccharomyces pombe, and has been shown to be involved in programmed replication fork stalling.

Identification of the molecular dysfunction caused by glutamate dehydrogenase S445L mutation responsible for hyperinsulinism/hyperammonemia.

Congenital hyperinsulinism/hyperammonemia (HI/HA) syndrome gives rise to unregulated protein-induced insulin secretion from pancreatic beta-cells, fasting hypoglycemia and elevated plasma ammonia levels. Mutations associated with HI/HA were identified in the Glud1 gene, encoding for glutamate dehydrogenase (GDH). We aimed at identifying the molecular causes of dysregulation in insulin secretion and ammonia production conferred by the most frequent HI/HA mutation Ser445Leu.

Capturing Structural Heterogeneity in Chromatin Fibers.

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.

Structure of centromere chromatin: from nucleosome to chromosomal architecture.

The centromere is essential for the segregation of chromosomes, as it serves as attachment site for microtubules to mediate chromosome segregation during mitosis and meiosis. In most organisms, the centromere is restricted to one chromosomal region that appears as primary constriction on the condensed chromosome and is partitioned into two chromatin domains: The centromere core is characterized by the centromere-specific histone H3 variant CENP-A (also called cenH3) and is required for specifying the centromere and for building the kinetochore complex during mitosis. This core region is generally flanked by pericentric heterochromatin, characterized by nucleosomes containing H3 methylated on lysine 9 (H3K9me) that are bound by heterochromatin proteins.

SHREC Silences Heterochromatin via Distinct Remodeling and Deacetylation Modules.

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.

Revisiting chromatin binding of the Arabidopsis UV-B photoreceptor UVR8.

Background: Plants perceive UV-B through the UV RESISTANCE LOCUS 8 (UVR8) photoreceptor and UVR8 activation leads to changes in gene expression such as those associated with UV-B acclimation and stress tolerance. Albeit functionally unrelated, UVR8 shows some homology with RCC1 (Regulator of Chromatin Condensation 1) proteins from non-plant organisms at the sequence level. These proteins act as guanine nucleotide exchange factors for Ran GTPases and bind chromatin via histones.

Panspecies small-molecule disruptors of heterochromatin-mediated transcriptional gene silencing.

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.

CRL4-like Clr4 complex in Schizosaccharomyces pombe depends on an exposed surface of Dos1 for heterochromatin silencing.

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).

Chd5 requires PHD-mediated histone 3 binding for tumor suppression.

Chromodomain Helicase DNA binding protein 5 (CHD5) is a tumor suppressor mapping to 1p36, a genomic region that is frequently deleted in human cancer. Although CHD5 belongs to the CHD family of chromatin-remodeling proteins, whether its tumor-suppressive role involves an interaction with chromatin is unknown. Here we report that Chd5 binds the unmodified N terminus of H3 through its tandem plant homeodomains (PHDs).

The Chp1-Tas3 core is a multifunctional platform critical for gene silencing by RITS.

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.

High-affinity binding of Chp1 chromodomain to K9 methylated histone H3 is required to establish centromeric heterochromatin.

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.

The molecular architecture of the metalloprotease FtsH.

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.

X-ray structure of a tetranucleosome and its implications for the chromatin fibre.

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.

Nucleosome arrays reveal the two-start organization of the chromatin fiber.

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.

Chromatin fiber folding: requirement for the histone H4 N-terminal tail.

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.