The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus

In sexually reproducing animals, a crucial step in zygote formation is the decondensation of the fertilizing sperm nucleus into a DNA replication-competent male pronucleus. Genome-wide nucleosome assembly on paternal DNA implies the replacement of sperm chromosomal proteins, such as protamines, by maternally provided histones. This fundamental process is specifically impaired in sésame (ssm), a unique Drosophila maternal effect mutant that prevents male pronucleus formation. Here we show that ssm is a point mutation in the Hira gene, thus demonstrating that the histone chaperone protein HIRA is required for nucleosome assembly during sperm nucleus decondensation. In vertebrates, HIRA has recently been shown to be critical for a nucleosome assembly pathway independent of DNA synthesis that specifically involves the H3.3 histone variant. We also show that nucleosomes containing H3.3, and not H3, are specifically assembled in paternal Drosophila chromatin before the first round of DNA replication. The exclusive marking of paternal chromosomes with H3.3 represents a primary epigenetic distinction between parental genomes in the zygote, and underlines an important consequence of the critical and highly specialized function of HIRA at fertilization.     

Dynamics of ATP-dependent chromatin assembly by ACF

The assembly of DNA into chromatin is a critical step in the replication and repair of the eukaryotic genome. It has been known for nearly 20 years that chromatin assembly is an ATP-dependent process. ATP-dependent chromatin-assembly factor (ACF) uses the energy of ATP hydrolysis for the deposition of histones into periodic nucleosome arrays, and the ISWI subunit of ACF is an ATPase that is related to helicases. Here we show that ACF becomes committed to the DNA template upon initiation of chromatin assembly. We also observed that ACF assembles nucleosomes in localized arrays, rather than randomly distributing them. By using a purified ACF-dependent system for chromatin assembly, we found that ACF hydrolyses about 2#150;4 molecules of ATP per base pair in the assembly of nucleosomes. This level of ATP hydrolysis is similar to that used by DNA helicases for the unwinding of DNA. These results suggest that a tracking mechanism exists in which ACF assembles chromatin as an ATP-driven DNA-translocating motor. Moreover, this proposed mechanism for ACF may be relevant to the function of other chromatin-remodelling factors that contain ISWI subunits.     

XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.     

盐透析体外组装核小体及检测方法

核小体是构成真核生物染色质的基本结构单位,体内研究核小体及染色质结构受到诸多因素限制,体外重构核小体结构是研究与核小体及染色质结构相关课题的一种重要的方法手段.实验将ES1,CS1以及601DNA序列克隆到载体中,通过PCR大量扩增回收得到目的DNA条带,表达纯化了4种组蛋白且装配成组蛋白八聚体,在盐透析的条件下组装形成核小体结构,利用EB染色以及Biotin标记的方法分析检测了形成核小体的效率.结果显示,在盐透析的条件下,可以有效的组装形成核小体结构,而且随着组蛋白八聚体与DNA的比例增加,核小体的形成效率显著提高.本实验为核小体定位、染色质重塑及组蛋白变体等表观遗传学以及结构生物学领域的研究奠定一定的基础.     

A role for cell-cycle-regulated histone H3 lysine 56 acetylation in the DNA damage response

DNA breaks are extremely harmful lesions that need to be repaired efficiently throughout the genome. However, the packaging of DNA into nucleosomes is a significant barrier to DNA repair, and the mechanisms of repair in the context of chromatin are poorly understood. Here we show that lysine 56 (K56) acetylation is an abundant modification of newly synthesized histone H3 molecules that are incorporated into chromosomes during S phase. Defects in the acetylation of K56 in histone H3 result in sensitivity to genotoxic agents that cause DNA strand breaks during replication. In the absence of DNA damage, the acetylation of histone H3 K56 largely disappears in G2. In contrast, cells with DNA breaks maintain high levels of acetylation, and the persistence of the modification is dependent on DNA damage checkpoint proteins. We suggest that the acetylation of histone H3 K56 creates a favourable chromatin environment for DNA repair and that a key component of the DNA damage response is to preserve this acetylation.     

Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA

The nucleosome subunits of chromatin are assembled from histones and DNA by an acidic protein which binds histones. The nucleosome assembly protein has been identified and purified from eggs of Xenopus laevis.     

In vitro centromere and kinetochore assembly on defined chromatin templates

During cell division, chromosomes are segregated to nascent daughter cells by attaching to the microtubules of the mitotic spindle through the kinetochore. Kinetochores are assembled on a specialized chromatin domain called the centromere, which is characterized by the replacement of nucleosomal histone H3 with the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in all eukaryotes but it is unknown how CENP-A chromatin directs centromere and kinetochore assembly. Here we generate synthetic CENP-A chromatin that recapitulates essential steps of centromere and kinetochore assembly in vitro. We show that reconstituted CENP-A chromatin when added to cell-free extracts is sufficient for the assembly of centromere and kinetochore proteins, microtubule binding and stabilization, and mitotic checkpoint function. Using chromatin assembled from histone H3/CENP-A chimaeras, we demonstrate that the conserved carboxy terminus of CENP-A is necessary and sufficient for centromere and kinetochore protein recruitment and function but that the CENP-A targeting domain--required for new CENP-A histone assembly--is not. These data show that two of the primary requirements for accurate chromosome segregation, the assembly of the kinetochore and the propagation of CENP-A chromatin, are specified by different elements in the CENP-A histone. Our unique cell-free system enables complete control and manipulation of the chromatin substrate and thus presents a powerful tool to study centromere and kinetochore assembly.