Transmission of modified nucleosomes from the mouse male germline to the zygote and subsequent remodeling of paternal chromatin

Rapidly after gamete fusion, the sperm nucleus loses its specific chromatin conformation and the DNA is repopulated with maternally derived nucleosomes. We evaluated the nature of paternally derived nucleosomes and the dynamics of sperm chromatin remodeling in the zygote directly after gamete fusion. We observed histone H4 acetylated at K8 or K12 already prior to full decondensation of the sperm nucleus, suggesting that these marks are transmitted by the spermatozoon. Tracking down the origin of H4K8ac and H4K12ac during spermiogenesis revealed the retention of nucleosomes with these modifications in the chromocenter of elongating spermatids. We show that sperm constitutive heterochromatin is enriched for nucleosomes carrying specific histone modifications which are transmitted to the zygote. Our results suggest an epigenetic mechanism for inheritance of chromosomal architecture. Furthermore, up to pronucleus formation, histone acetylation and phosphorylation build up in a cascade-like fashion in the paternal chromatin. After formation of the pronucleus, a subset of these marks is removed from the heterochromatin, which suggests a reestablishment of the euchromatin-heterochromatin partition.     

The Snf2 Homolog Fun30 Acts as a Homodimeric ATP-dependent Chromatin-remodeling Enzyme

The Saccharomyces cerevisiae Fun30 (Function unknown now 30) protein shares homology with an extended family of Snf2-related ATPases. Here we report the purification of Fun30 principally as a homodimer with a molecular mass of about 250 kDa. Biochemical characterization of this complex reveals that it has ATPase activity stimulated by both DNA and chromatin. Consistent with this, it also binds to both DNA and chromatin. The Fun30 complex also exhibits activity in ATP-dependent chromatin remodeling assays. Interestingly, its activity in histone dimer exchange is high relative to the ability to reposition nucleosomes. Fun30 also possesses a weakly conserved CUE motif suggesting that it may interact specifically with ubiquitinylated proteins. However, in vitro Fun30 was found to have no specificity in its interaction with ubiquitinylated histones.     

Functions of chromatin remodeling factors in heterochromatin formation and maintenance

Heterochromatin is characteristically more compact than euchromatin in the eukaryotic genome. The establishment of heterochromatin is mediated by special histone modifications, recruitment and propagation of heterochromatin specific proteins, as well as formation of special primary and high order structures of chromatin. Chromatin remodeling factors are ATPases that can alter the conformation and/or positioning of nucleosomes along DNA in an ATP-dependent manner. There is increasing evidence implicating chromatin remodeling activities in heterochromatin in various organisms ranging from yeasts to humans. Chromatin remodeling factors play roles in the establishment, maintenance and epigenetic inheritance of heterochromatin, but the underlying molecular mechanisms have just begun to be investigated.     

Histone modifications influence the action of Snf2 family remodelling enzymes by different mechanisms

Alteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes.     

Mechanisms of HP1-mediated gene silencing in Drosophila

Heterochromatin Protein 1 (HP1) is a structural component of silent chromatin at telomeres and centromeres. Euchromatic genes repositioned near heterochromatin by chromosomal rearrangements are typically silenced in an HP1-dependent manner. Silencing is thought to involve the spreading of heterochromatin proteins over the rearranged genes. HP1 associates with centric heterochromatin through an interaction with methylated lysine 9 of histone H3, a modification generated by SU(VAR)3-9. The current model for spreading of silent chromatin involves HP1-dependent recruitment of SU(VAR)3-9, resulting in the methylation of adjacent nucleosomes and association of HP1 along the chromatin fiber. To address mechanisms of silent chromatin formation and spreading, HP1 was fused to the DNA-binding domain of the E. coli lacI repressor and expressed in Drosophila melanogaster stocks carrying heat shock reporter genes positioned 1.9 and 3.7 kb downstream of lac operator repeats. Association of lacI-HP1 with the repeats resulted in silencing of both reporter genes and correlated with a closed chromatin structure consisting of regularly spaced nucleosomes, similar to that observed in centric heterochromatin. Chromatin immunoprecipitation experiments demonstrated that HP1 spread bi-directionally from the tethering site and associated with the silenced reporter transgenes. To examine mechanisms of spreading, the effects of a mutation in Su(var)3-9 were investigated. Silencing was minimally affected at 1.9 kb, but eliminated at 3.7 kb, suggesting that HP1-mediated silencing can operate in a SU(VAR)3-9-independent and -dependent manner.     

Two distinct mechanisms of chromatin interaction by the Isw2 chromatin remodeling complex in vivo

We have previously shown that Saccharomyces cerevisiae Isw2 complex slides nucleosomes to remodel chromatin in vivo. Our data suggested a model in which Isw2 complex binds the histone octamer and DNA separately to generate the force necessary for nucleosome movement. Here we find that the histone H4 "basic patch" is the only portion of any amino-terminal histone tail required for both target-specific association of Isw2 complex with chromatin and chromatin remodeling in vivo, whereas it is dispensable for basal levels of chromatin binding. Similarly, we find that nonremodeled chromatin structure and integrity of Isw2 complex are required only for target-specific association of Isw2 with chromatin. These data demonstrate fundamental differences between the target-specific and basal modes of chromatin binding by Isw2 complex in vivo and suggest that only the former involves contributions from DNA, histone H4, and sequence-specific DNA binding proteins. We propose a model for target recognition and chromatin remodeling by Isw2 complex in vivo.     

Analysis of chromatin structure of genes silenced by heterochromatin in trans

While heterochromatic gene silencing in cis is often accompanied by nucleosomal compaction, characteristic histone modifications, and recruitment of heterochromatin proteins, little is known concerning genes silenced by heterochromatin in trans. An insertion of heterochromatic satellite DNA in the euchromatic brown (bw) gene of Drosophila melanogaster results in bwDominant (bwD), which can inactivate loci on the homolog by relocation near the centric heterochromatin (trans-inactivation). Nucleosomal compaction was found to accompany trans-inactivation, but stereotypical heterochromatic histone modifications were mostly absent on silenced reporter genes. HP1 was enriched on trans-inactivated reporter constructs and this enrichment was more pronounced on adult chromatin than on larval chromatin. Interestingly, this HP1 enrichment in trans was unaccompanied by an increase in the 2MeH3K9 mark, which is generally thought to be the docking site for HP1 in heterochromatin. However, a substantial increase in the 2MeH3K9 mark was found on or near the bwD satellite insertion in cis, but did not spread further. These observations suggest that the interaction of HP1 with chromatin in cis is fundamentally different from that in trans. Our molecular data agree well with the differential phenotypic effect on bwD trans-inactivation of various genes known to be involved in histone modification and cis gene silencing.     

Antagonistic forces that position nucleosomes in vivo

ATP-dependent chromatin remodeling complexes are implicated in many areas of chromosome biology. However, the physiological role of many of these enzymes is still unclear. In budding yeast, the Isw2 complex slides nucleosomes along DNA. By analyzing the native chromatin structure of Isw2 targets, we have found that nucleosomes adopt default, DNA-directed positions when ISW2 is deleted. We provide evidence that Isw2 targets contain DNA sequences that are inhibitory to nucleosome formation and that these sequences facilitate the formation of nuclease-accessible open chromatin in the absence of Isw2. Our data show that the biological function of Isw2 is to position nucleosomes onto unfavorable DNA. These results reveal that antagonistic forces of Isw2 and the DNA sequence can control nucleosome positioning and genomic access in vivo.     

Analysis of nucleosome repositioning by yeast ISWI and Chd1 chromatin remodeling complexes

ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin remodeling activities in eukaryotes from yeast to man. Many of these complexes have been found to reposition nucleosomes but with different directionalities. We find that the yeast Isw1a, Isw2, and Chd1 enzymes preferentially move nucleosomes toward more central locations on short DNA fragments whereas Isw1b does not. Importantly, the inherent positioning properties of the DNA play an important role in determining where nucleosomes are relocated to by all of these enzymes. However, a key difference is that the Isw1a, Isw2, and Chd1 enzymes are unable to move nucleosomes to positions closer than 15 bp from a DNA end, whereas Isw1b can. We also find that there is a correlation between the inability of enzymes to move nucleosomes close to DNA ends and the preferential binding to nucleosomes bearing linker DNA. These observations suggest that the accessibility of linker DNA together with the positioning properties of the underlying DNA play important roles in determining the outcome of remodeling by these enzymes.