Chromatin localization of nucleophosmin organizes ribosome biogenesis.

Ribosome biogenesis takes place in the nucleolus, a nuclear membrane-less organelle. Although well studied, it remains unknown how nascent ribosomal subunits separate from the central chromatin compartment and move to the outer granular component, where maturation occurs. We find that the Schizosaccharomyces pombe nucleophosmin-like protein Fkbp39 localizes to rDNA sites encoding the 60S subunit rRNA, and this localization contributes to its specific association with nascent 60S subunits. Fkbp39 dissociates from chromatin to bind nascent 60S subunits, causing the latter to partition away from chromatin and from nascent 40S subunits through liquid-liquid phase separation. In vivo, Fkbp39 binding directs the translocation of nascent 60S subunits toward the nucleophosmin-rich granular component. This process increases the efficiency of 60S subunit assembly, facilitating the incorporation of 60S RNA domain III. Thus, chromatin localization determines the specificity of nucleophosmin in sorting nascent ribosomal subunits and coordinates their movement into specialized assembly compartments within the nucleolus.

Histone modifications regulate pioneer transcription factor cooperativity.

Pioneer transcription factors have the ability to access DNA in compacted chromatin1. Multiple transcription factors can bind together to a regulatory element in a cooperative way, and cooperation between the pioneer transcription factors OCT4 (also known as POU5F1) and SOX2 is important for pluripotency and reprogramming2-4. However, the molecular mechanisms by which pioneer transcription factors function and cooperate on chromatin remain unclear. Here we present cryo-electron microscopy structures of human OCT4 bound to a nucleosome containing human LIN28B or nMATN1 DNA sequences, both of which bear multiple binding sites for OCT4. Our structural and biochemistry data reveal that binding of OCT4 induces changes to the nucleosome structure, repositions the nucleosomal DNA and facilitates cooperative binding of additional OCT4 and of SOX2 to their internal binding sites. The flexible activation domain of OCT4 contacts the N-terminal tail of histone H4, altering its conformation and thus promoting chromatin decompaction. Moreover, the DNA-binding domain of OCT4 engages with the N-terminal tail of histone H3, and post-translational modifications at H3K27 modulate DNA positioning and affect transcription factor cooperativity. Thus, our findings suggest that the epigenetic landscape could regulate OCT4 activity to ensure proper cell programming.

Fuzzy Interactions Form and Shape the Histone Transport Complex.

Protein transport into the nucleus is mediated by transport receptors. Import of highly charged proteins, such as histone H1 and ribosomal proteins, requires a dimer of two transport receptors. In this study, we determined the cryo-EM structure of the Imp7:Impß:H1.0 complex, showing that the two importins form a cradle that accommodates the linker histone. The H1.0 globular domain is bound to Impß, whereas the acidic loops of Impß and Imp7 chaperone the positively charged C-terminal tail. Although it remains disordered, the H1 tail serves as a zipper that closes and stabilizes the structure through transient non-specific interactions with importins. Moreover, we found that the GGxxF and FxFG motifs in the Imp7 C-terminal tail are essential for Imp7:Impß dimerization and H1 import, resembling importin interaction with nucleoporins, which, in turn, promote complex disassembly. The architecture of many other complexes might be similarly defined by rapidly exchanging electrostatic interactions mediated by disordered regions.

Bridging of DNA breaks activates PARP2-HPF1 to modify chromatin.

Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify histones and other substrates through the addition of mono- and poly(ADP-ribose) (PAR)1-5. In the DNA damage responses, this post-translational modification occurs predominantly on serine residues6-8 and requires HPF1, an accessory factor that switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine9,10. Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin decompaction and provides an anchor for the recruitment of downstream signalling and repair factors to the sites of DNA breaks2,11. Here, to understand the molecular mechanism by which PARP enzymes recognize DNA breaks within chromatin, we determined the cryo-electron-microscopic structure of human PARP2-HPF1 bound to a nucleosome. This showed that PARP2-HPF1 bridges two nucleosomes, with the broken DNA aligned in a position suitable for ligation, revealing the initial step in the repair of double-strand DNA breaks. The bridging induces structural changes in PARP2 that signal the recognition of a DNA break to the catalytic domain, which licenses HPF1 binding and PARP2 activation. Our data suggest that active PARP2 cycles through different conformational states to exchange NAD+ and substrate, which may enable PARP enzymes to act processively while bound to chromatin. The processes of PARP activation and the PARP catalytic cycle we describe can explain mechanisms of resistance to PARP inhibitors and will aid the development of better inhibitors as cancer treatments12-16.

Dicer-independent primal RNAs trigger RNAi and heterochromatin formation.

Assembly of fission yeast pericentromeric heterochromatin and generation of small interfering RNAs (siRNAs) from noncoding centromeric transcripts are mutually dependent processes. How this interdependent positive feedback loop is first triggered is a fundamental unanswered question. Here, we show that two distinct Argonaute (Ago1)-dependent pathways mediate small RNA generation. RNA-dependent RNA polymerase complex (RDRC) and Dicer act on specific noncoding RNAs to generate siRNAs by a mechanism that requires the slicer activity of Ago1 but is independent of pre-existing heterochromatin. In the absence of RDRC or Dicer, a distinct class of small RNAs, called primal small RNAs (priRNAs), associates with Ago1. priRNAs are degradation products of abundant transcripts, which bind to Ago1 and target antisense transcripts that result from bidirectional transcription of DNA repeats. Our results suggest that a transcriptome surveillance mechanism based on random association of RNA degradation products with Argonaute triggers siRNA amplification and heterochromatin assembly within DNA repeats.

Transposon silencing by piRNAs.

In animal cells, small RNA molecules, called piRNAs, defend the genome against selfish DNA elements such as transposons. In this issue, Klattenhoff et al. (2009) report that an HP1 family protein, Rhino, is required for piRNA generation and transposon silencing in Drosophila germline cells. The results provide a link between heterochromatin and piRNA-mediated genome defense.

Ccr4-Not complex reduces transcription efficiency in heterochromatin.

Heterochromatic silencing is thought to occur through a combination of transcriptional silencing and RNA degradation, but the relative contribution of each pathway is not known. In this study, we analyzed RNA Polymerase II (RNA Pol II) occupancy and levels of nascent and steady-state RNA in different mutants of Schizosaccharomyces pombe, in order to quantify the contribution of each pathway to heterochromatic silencing. We found that transcriptional silencing consists of two components, reduced RNA Pol II accessibility and, unexpectedly, reduced transcriptional efficiency. Heterochromatic loci showed lower transcriptional output compared to euchromatic loci, even when comparable amounts of RNA Pol II were present in both types of regions. We determined that the Ccr4-Not complex and H3K9 methylation are required for reduced transcriptional efficiency in heterochromatin and that a subset of heterochromatic RNA is degraded more rapidly than euchromatic RNA. Finally, we quantified the contribution of different chromatin modifiers, RNAi and RNA degradation to each silencing pathway. Our data show that several pathways contribute to heterochromatic silencing in a locus-specific manner and reveal transcriptional efficiency as a new mechanism of silencing.

CENP-C unwraps the human CENP-A nucleosome through the H2A C-terminal tail.

Centromeres are defined epigenetically by nucleosomes containing the histone H3 variant CENP-A, upon which the constitutive centromere-associated network of proteins (CCAN) is built. CENP-C is considered to be a central organizer of the CCAN. We provide new molecular insights into the structure of human CENP-A nucleosomes, in isolation and in complex with the CENP-C central region (CENP-CCR ), the main CENP-A binding module of human CENP-C. We establish that the short αN helix of CENP-A promotes DNA flexibility at the nucleosome ends, independently of the sequence it wraps. Furthermore, we show that, in vitro, two regions of human CENP-C (CENP-CCR and CENP-Cmotif ) both bind exclusively to the CENP-A nucleosome. We find CENP-CCR to bind with high affinity due to an extended hydrophobic area made up of CENP-AV532 and CENP-AV533 . Importantly, we identify two key conformational changes within the CENP-A nucleosome upon CENP-C binding. First, the loose DNA wrapping of CENP-A nucleosomes is further exacerbated, through destabilization of the H2A C-terminal tail. Second, CENP-CCR rigidifies the N-terminal tail of H4 in the conformation favoring H4K20 monomethylation, essential for a functional centromere.

Shelterin and subtelomeric DNA sequences control nucleosome maintenance and genome stability.

Telomeres and the shelterin complex cap and protect the ends of chromosomes. Telomeres are flanked by the subtelomeric sequences that have also been implicated in telomere regulation, although their role is not well defined. Here, we show that, in Schizosaccharomyces pombe, the telomere-associated sequences (TAS) present on most subtelomeres are hyper-recombinogenic, have metastable nucleosomes, and unusual low levels of H3K9 methylation. Ccq1, a subunit of shelterin, protects TAS from nucleosome loss by recruiting the heterochromatic repressor complexes CLRC and SHREC, thereby linking nucleosome stability to gene silencing. Nucleosome instability at TAS is independent of telomeric repeats and can be transmitted to an intrachromosomal locus containing an ectopic TAS fragment, indicating that this is an intrinsic property of the underlying DNA sequence. When telomerase recruitment is compromised in cells lacking Ccq1, DNA sequences present in the TAS promote recombination between chromosomal ends, independent of nucleosome abundance, implying an active function of these sequences in telomere maintenance. We propose that Ccq1 and fragile subtelomeres co-evolved to regulate telomere plasticity by controlling nucleosome occupancy and genome stability.

Argonaute and Triman generate dicer-independent priRNAs and mature siRNAs to initiate heterochromatin formation.

RNAi is a conserved mechanism in which small RNAs induce silencing of complementary targets. We have previously identified priRNAs, a class of Dicer-independent small RNAs in fission yeast. The mechanism by which Dicer-independent small RNAs are generated is not well understood in any species. Here we reconstitute the final steps of priRNA and siRNA biogenesis in vitro. We identify the 3'-5' exonuclease Triman and demonstrate that Argonaute, loaded with longer RNA precursors, recruits Triman to generate mature priRNAs and siRNAs. We show that priRNA and siRNA trimming is required for de novo assembly of heterochromatin at centromeric repeats and the mat locus and for maintenance of heterochromatin at developmental genes. Furthermore, in rrp6Δ cells RNAi targets diverse genes in a Triman-dependent way, indicating that the exosome protects the genome from spurious RNAi. Our results suggest that Argonaute association with RNA degradation products generates priRNAs and triggers RNAi in a process of transcriptome surveillance.

Disordered region of H3K9 methyltransferase Clr4 binds the nucleosome and contributes to its activity.

Heterochromatin is a distinctive chromatin structure that is essential for chromosome segregation, genome stability and regulation of gene expression. H3K9 methylation (H3K9me), a hallmark of heterochromatin, is deposited by the Su(var)3-9 family of proteins; however, the mechanism by which H3K9 methyltransferases bind and methylate the nucleosome is poorly understood. In this work we determined the interaction of Clr4, the fission yeast H3K9 methyltransferase, with nucleosomes using nuclear magnetic resonance, biochemical and genetic assays. Our study shows that the Clr4 chromodomain binds the H3K9me3 tail and that both, the chromodomain and the disordered region connecting the chromodomain and the SET domain, bind the nucleosome core. We show that interaction of the disordered region with the nucleosome core is independent of H3K9me and contributes to H3K9me in vitro and in vivo. Moreover, we show that those interactions with the nucleosome core are contributing to de novo deposition of H3K9me and to establishment of heterochromatin.

Accumulation of RNA on chromatin disrupts heterochromatic silencing.

Long noncoding RNAs (lncRNAs) play a conserved role in regulating gene expression, chromatin dynamics, and cell differentiation. They serve as a platform for RNA interference (RNAi)-mediated heterochromatin formation or DNA methylation in many eukaryotic organisms. We found in Schizosaccharomyces pombe that heterochromatin is lost at transcribed regions in the absence of RNA degradation. We show that heterochromatic RNAs are retained on chromatin, form DNA:RNA hybrids, and need to be degraded by the Ccr4-Not complex or RNAi to maintain heterochromatic silencing. The Ccr4-Not complex is localized to chromatin independently of H3K9me and degrades chromatin-associated transcripts, which is required for transcriptional silencing. Overexpression of heterochromatic RNA, but not euchromatic RNA, leads to chromatin localization and loss of silencing of a distant ade6 reporter in wild-type cells. Our results demonstrate that chromatin-bound RNAs disrupt heterochromatin organization and need to be degraded in a process of heterochromatin formation.

The methyltransferase activity of Clr4Suv39h triggers RNAi independently of histone H3K9 methylation.

In fission yeast, the pericentromeric dg and dh repeats are transcribed and give rise to small interfering RNAs (siRNAs) by a mechanism that depends on the Clr4(suv39h) histone H3 lysine 9 (H3K9) methyltransferase. Here, we show that Clr4 activity promotes the assembly of a tripartite complex composed of the Clr4-containing CLRC complex and complexes involved in siRNA generation. However, unlike dh siRNAs, dg siRNAs accumulate to near wild-type levels in cells with H3K9 substitutions that cannot be methylated. Thus, Clr4 activity controls siRNA amplification from the different repeat regions by different mechanisms, H3K9 methylation dependent versus independent. Furthermore, artificial tethering of Rik1, a core subunit of the CLRC complex, to a euchromatic RNA mediates RNAi-dependent silencing that partially bypasses the requirement for other CLRC subunits. These findings establish Rik1 as a key link between CLRC and RNAi and reveal distinct centromeric siRNA amplification mechanisms that depend on the Clr4 methyltransferase activity.

22G-RNAs in transposon silencing and centromere function.

Three recent papers (Gu et al., 2009; Claycomb et al., 2009; van Wolfswinkel et al., 2009) provide evidence that links a new class of small RNAs and Argonaute-associated complexes to centromere function and genome surveillance.

ISWI catalyzes nucleosome sliding in condensed nucleosome arrays.

How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. Here we investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner, qualitatively agree with our data. We speculate that monkey-bar mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.

Histone modifications regulate pioneer transcription factor binding and cooperativity.

Pioneer transcription factors have the ability to access DNA in compacted chromatin. Multiple transcription factors can bind together to a regulatory element in a cooperative way and cooperation between pioneer transcription factors Oct4 and Sox2 is important for pluripotency and reprogramming. However, the molecular mechanisms by which pioneer transcription factors function and cooperate remain unclear. Here we present cryo-EM structures of human Oct4 bound to a nucleosome containing human Lin28B and nMatn1 DNA sequences, which bear multiple binding sites for Oct4. Our structural and biochemistry data reveal that Oct4 binding induces changes to the nucleosome structure, repositions the nucleosomal DNA and facilitates cooperative binding of additional Oct4 and of Sox2 to their internal binding sites. The flexible activation domain of Oct4 contacts the histone H4 N-terminal tail, altering its conformation and thus promoting chromatin decompaction. Moreover, the DNA binding domain of Oct4 engages with histone H3 N-terminal tail, and posttranslational modifications at H3K27 modulate DNA positioning and affect transcription factor cooperativity. Thus, our data show that the epigenetic landscape can regulate Oct4 activity to ensure proper cell reprogramming.

CENP-A and CENP-B collaborate to create an open centromeric chromatin state.

Centromeres are epigenetically defined via the presence of the histone H3 variant CENP-A. Contacting CENP-A nucleosomes, the constitutive centromere associated network (CCAN) and the kinetochore assemble, connecting the centromere to spindle microtubules during cell division. The DNA-binding centromeric protein CENP-B is involved in maintaining centromere stability and, together with CENP-A, shapes the centromeric chromatin state. The nanoscale organization of centromeric chromatin is not well understood. Here, we use single-molecule fluorescence and cryoelectron microscopy (cryoEM) to show that CENP-A incorporation establishes a dynamic and open chromatin state. The increased dynamics of CENP-A chromatin create an opening for CENP-B DNA access. In turn, bound CENP-B further opens the chromatin fiber structure and induces nucleosomal DNA unwrapping. Finally, removal of CENP-A increases CENP-B mobility in cells. Together, our studies show that the two centromere-specific proteins collaborate to reshape chromatin structure, enabling the binding of centromeric factors and establishing a centromeric chromatin state.

ISWI catalyzes nucleosome sliding in condensed nucleosome arrays.

How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. We investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner qualitatively agree with our data. We speculate that 'monkey-bar' mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.

Following the signal sequence from ribosomal tunnel exit to signal recognition particle.

Membrane and secretory proteins can be co-translationally inserted into or translocated across the membrane. This process is dependent on signal sequence recognition on the ribosome by the signal recognition particle (SRP), which results in targeting of the ribosome-nascent-chain complex to the protein-conducting channel at the membrane. Here we present an ensemble of structures at subnanometre resolution, revealing the signal sequence both at the ribosomal tunnel exit and in the bacterial and eukaryotic ribosome-SRP complexes. Molecular details of signal sequence interaction in both prokaryotic and eukaryotic complexes were obtained by fitting high-resolution molecular models. The signal sequence is presented at the ribosomal tunnel exit in an exposed position ready for accommodation in the hydrophobic groove of the rearranged SRP54 M domain. Upon ribosome binding, the SRP54 NG domain also undergoes a conformational rearrangement, priming it for the subsequent docking reaction with the NG domain of the SRP receptor. These findings provide the structural basis for improving our understanding of the early steps of co-translational protein sorting.

Structure of eEF3 and the mechanism of transfer RNA release from the E-site.

Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of the aminoacyl-tRNA-eEF1A-GTP ternary complex to the ribosomal A-site and has been suggested to facilitate the clearance of deacyl-tRNA from the E-site. Here we present the crystal structure of Saccharomyces cerevisiae eEF3, showing that it consists of an amino-terminal HEAT repeat domain, followed by a four-helix bundle and two ABC-type ATPase domains, with a chromodomain inserted in ABC2. Moreover, we present the cryo-electron microscopy structure of the ATP-bound form of eEF3 in complex with the post-translocational-state 80S ribosome from yeast. eEF3 uses an entirely new factor binding site near the ribosomal E-site, with the chromodomain likely to stabilize the ribosomal L1 stalk in an open conformation, thus allowing tRNA release.