Nucleosomes Stabilize ssRNA-dsDNA Triple Helices in Human Cells

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The right place at the right time: chaperoning core histone variants

Histone proteins dynamically regulate chromatin structure and epigenetic signaling to maintain cell homeostasis. These processes require controlled spatial and temporal deposition and eviction of histones by their dedicated chaperones. With the evolution of histone variants, a network of functionally specific histone chaperones has emerged. Molecular details of the determinants of chaperone specificity for different histone variants are only slowly being resolved. A complete understanding of these processes is essential to shed light on the genuine biological roles of histone variants, their chaperones, and their impact on chromatin dynamics.     

A Comparison of In Vitro Nucleosome Positioning Mapped with Chicken,Frog and a Variety of Yeast Core Histones

Using high-throughput sequencing, we have mapped sequence-directed nucleosome positioning in vitro on four plasmid DNAs containing DNA fragments derived from the genomes of sheep, drosophila, human and yeast. Chromatins were prepared by reconstitution using chicken, frog and yeast core histones. We also assembled yeast chromatin in which histone H3 was replaced by the centromere-specific histone variant, Cse4. The positions occupied by recombinant frog and native chicken histones were found to be very similar. In contrast, nucleosomes containing the canonical yeast octamer or, in particular, the Cse4 octamer were assembled at distinct populations of locations, a property that was more apparent on particular genomic DNA fragments. The factors that may contribute to this variation in nucleosome positioning and the implications of the behavior are discussed.     

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. 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.     

dBigH1, a second histone H1 in Drosophila,and the consequences for histone fold nomenclature

Recently, Pérez-Montero and colleagues (Developmental cell, 26: 578–590, 2013) described the occurrence of a new histone H1 variant (dBigH1) in Drosophila. The presence of unusual acidic amino acid patches at the N-terminal end of dBigH1 is in contrast to the arginine patches that exist at the N- and C-terminal domains of other histone H1-related proteins found in the sperm of some organisms. This departure from the strictly lysine-rich composition of the somatic histone H1 raises a question about the true definition of its protein members. Their minimal essential requirements appear to be the presence of a lysine- and alanine–rich, intrinsically disordered C-terminal domain, with a highly helicogenic potential upon binding to the linker DNA regions of chromatin. In metazoans, specific targeting of these regions is further achieved by a linker histone fold domain (LHFD), distinctively different from the characteristic core histone fold domain (CHFD) of the nucleosome core histones.     

Disruption of NAP1 genes in Arabidopsis thaliana suppresses the fas1 mutant phenotype,enhances genome stability and changes chromatin compaction

Histone chaperones mediate the assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. In Arabidopsis thaliana, loss-of-function of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1 (CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. CAF-1 dysfunction can lead to altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage, clearly demonstrating its severity. How chromatin composition is maintained without functional CAF-1 remains elusive. Here we show that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) suppresses the FAS1 loss-of-function phenotype. The quadruple mutant fas1 nap1;1 nap1;2 nap1;3 shows wild-type growth, decreased sensitivity to genotoxic stress and suppression of telomere and 45S rDNA loss. Chromatin of fas1 nap1;1 nap1;2 nap1;3 plants is less accessible to micrococcal nuclease and the nuclear H3.1 and H3.3 histone pools change compared to fas1. Consistently, association between NAP1 and H3 occurs in the cytoplasm and nucleus in vivo in protoplasts. Altogether we show that NAP1 proteins play an essential role in DNA repair in fas1, which is coupled to nucleosome assembly through modulation of H3 levels in the nucleus.     

dBigH1, a second histone H1 in Drosophila,and the consequences for histone fold nomenclature

Recently, Pérez-Montero and colleagues (Developmental cell, 26: 578–590, 2013) described the occurrence of a new histone H1 variant (dBigH1) in Drosophila. The presence of unusual acidic amino acid patches at the N-terminal end of dBigH1 is in contrast to the arginine patches that exist at the N- and C-terminal domains of other histone H1-related proteins found in the sperm of some organisms. This departure from the strictly lysine-rich composition of the somatic histone H1 raises a question about the true definition of its protein members. Their minimal essential requirements appear to be the presence of a lysine- and alanine–rich, intrinsically disordered C-terminal domain, with a highly helicogenic potential upon binding to the linker DNA regions of chromatin. In metazoans, specific targeting of these regions is further achieved by a linker histone fold domain (LHFD), distinctively different from the characteristic core histone fold domain (CHFD) of the nucleosome core histones.     

依赖ATP的染色质物理修饰

染色质重塑是基因表达调控过程中一个非常重要的环节 .染色质重塑主要包括 2种类型 :一种是依赖ATP的物理修饰 ,另一种是依赖共价结合反应的化学修饰 .依赖ATP的物理修饰主要是利用ATP水解释放的能量 ,使DNA超螺旋旋矩和旋相发生变化 ,使转录因子更易接近并结合核小体DNA ,从而调控基因的转录过程     

Specific contributions of histone tails and their acetylation to the mechanical stability of nucleosomes

The distinct contributions of histone tails and their acetylation to nucleosomal stability were examined by mechanical disruption of individual nucleosomes in a single chromatin fiber using an optical trap. Enzymatic removal of H2A/H2B tails primarily decreased the strength of histone-DNA interactions located approximately +/-36bp from the dyad axis of symmetry (off-dyad strong interactions), whereas removal of the H3/H4 tails played a greater role in regulating the total amount of DNA bound. Similarly, nucleosomes composed of histones acetylated to different degrees by the histone acetyltransferase p300 exhibited significant decreases in the off-dyad strong interactions and the total amount of DNA bound. Acetylation of H2A/H2B appears to play a particularly critical role in weakening the off-dyad strong interactions. Collectively, our results suggest that the destabilizing effects of tail acetylation may be due to elimination of specific key interactions in the nucleosome.     

Nucleosome dyad determines the H1 C-terminus collapse on distinct DNA arms

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组蛋白修饰在染色质复制过程中的作用

真核生物中的DNA复制,不但要保证DNA编码的基因组信息高保真复制,也要保证染色质结构所蕴含的表观遗传组稳定传递,这个过程对于维持基因组的完整性和稳定性至关重要。时至今日,人们对DNA复制的机制已经有了深入的认识,但是对染色质复制以及表观遗传信息传递的了解才刚刚开始。组蛋白是染色质结构中最主要的蛋白组成部分,其上面丰富的转录后修饰是表观遗传调控的核心方式之一。从最近几年组蛋白的修饰研究进展入手,主要综述在DNA复制过程中组蛋白修饰如何参与染色质复制的调控。     

Evidence for the nuclear import of histones H3.1 and H4 as monomers

Newly synthesised histones are thought to dimerise in the cytosol and undergo nuclear import in complex with histone chaperones. Here, we provide evidence that human H3.1 and H4 are imported into the nucleus as monomers. Using a tether‐and‐release system to study the import dynamics of newly synthesised histones, we find that cytosolic H3.1 and H4 can be maintained as stable monomeric units. Cytosolically tethered histones are bound to importin‐alpha proteins (predominantly IPO4), but not to histone‐specific chaperones NASP, ASF1a, RbAp46 (RBBP7) or HAT1, which reside in the nucleus in interphase cells. Release of monomeric histones from their cytosolic tether results in rapid nuclear translocation, IPO4 dissociation and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones reveals an excess of H3 specifically associated with sNASP, suggesting that NASP maintains a soluble, monomeric pool of H3 within the nucleus and may act as a nuclear receptor for newly imported histone. In summary, we propose that histones H3 and H4 are rapidly imported as monomeric units, forming heterodimers in the nucleus rather than the cytosol.     

Regulation of histone H3K4 methylation in brain development and disease

The growing list of mutations implicated in monogenic disorders of the developing brain includes at least seven genes (ARX, CUL4B, KDM5A, KDM5C, KMT2A, KMT2C, KMT2D) with loss-of-function mutations affecting proper regulation of histone H3 lysine 4 methylation, a chromatin mark which on a genome-wide scale is broadly associated with active gene expression, with its mono-, di- and trimethylated forms differentially enriched at promoter and enhancer and other regulatory sequences. In addition to these rare genetic syndromes, dysregulated H3K4 methylation could also play a role in the pathophysiology of some cases diagnosed with autism or schizophrenia, two conditions which on a genome-wide scale are associated with H3K4 methylation changes at hundreds of loci in a subject-specific manner. Importantly, the reported alterations for some of the diseased brain specimens included a widespread broadening of H3K4 methylation profiles at gene promoters, a process that could be regulated by the UpSET(KMT2E/MLL5)-histone deacetylase complex. Furthermore, preclinical studies identified maternal immune activation, parental care and monoaminergic drugs as environmental determinants for brain-specific H3K4 methylation. These novel insights into the epigenetic risk architectures of neurodevelopmental disease will be highly relevant for efforts aimed at improved prevention and treatment of autism and psychosis spectrum disorders.     

Nucleosomes in solution exist as a mixture of twist-defect states

The 2.0 A crystal structure of a nucleosome core particle in complex with a bivalent pyrrole-imidazole polyamide reveals that this "clamp" effectively crossbraces the two gyres of the DNA superhelix, thereby stabilizing the nucleosome against dissociation. Using X-ray crystallography and footprinting techniques, we show that the clamp preferentially binds nucleosomes over free DNA, and that nucleosomal DNA exists as a mixture of multiple twist-defect intermediates in solution. The nucleosomes exist in one of two different conformations in various crystal structures that trap twist-defect intermediates, even on a strong positioning sequence. Evidence has been obtained supporting the existence of twist-defect states in nucleosomal DNA in solution that are similar to those obtained in crystal structures. Our results also substantiate the idea that twist diffusion may represent an important means of altering the accessibility of nucleosomal DNA both in the presence and in the absence of ATP-dependent chromatin-remodelling enzymes.