The effect of histone hyperacetylation on the nuclease sensitivity and the solubility of chromatin

We have examined the effects of histone hyperacetylation upon nuclease digestion of nuclei and subsequent fractionation of chromosomal material in the presence of MgCl2. DNase I shows a maximum sensitivity towards hyperacetylated nuclei at somewhat elevated ionic strengths (150-200 mM NaCl), whereas micrococcal nuclease exhibits no specificity for acetylated nuclei over a broad range of ionic strengths. Fractionation in the presence of MgCl2 of hyperacetylated nuclei digested with micrococcal nuclease results in a substantial increase in the amount of soluble chromatin relative to that obtained with control nuclei. This increased yield of Mg2+-soluble chromatin results from the recruitment into this fraction of oligonucleosomes containing extremely hyperacetylated histones. These results suggest that contiguous nucleosomes containing highly acetylated histones may be altered in their ability to interact with themselves and with other nucleosomes.     

Combinatorial chromatin modification patterns in the human genome revealed by subspace clustering

Chromatin modifications, such as post-translational modification of histone proteins and incorporation of histone variants, play an important role in regulating gene expression. Joint analyses of multiple histone modification maps are starting to reveal combinatorial patterns of modifications that are associated with functional DNA elements, providing support to the 'histone code' hypothesis. However, due to the lack of analytical methods, only a small number of chromatin modification patterns have been discovered so far. Here, we introduce a scalable subspace clustering algorithm, coherent and shifted bicluster identification (CoSBI), to exhaustively identify the set of combinatorial modification patterns across a given epigenome. Performance comparisons demonstrate that CoSBI can generate biclusters with higher intra-cluster coherency and biological relevance. We apply our algorithm to a compendium of 39 genome-wide chromatin modification maps in human CD4(+) T cells. We identify 843 combinatorial patterns that recur at >0.1% of the genome. A total of 19 chromatin modifications are observed in the combinatorial patterns, 10 of which occur in more than half of the patterns. We also identify combinatorial modification signatures for eight classes of functional DNA elements. Application of CoSBI to epigenome maps of different cells and developmental stages will aid in understanding how chromatin structure helps regulate gene expression.     

Eukaryote-conserved histone post-translational modification landscape in Giardia duodenalis revealed by mass spectrometry

Diarrheal disease caused by Giardia duodenalis is highly prevalent, causing over 200 million cases globally each year. The processes that drive parasite virulence, host immune evasion and transmission involve coordinated gene expression and have been linked to epigenetic regulation. Epigenetic regulatory systems are eukaryote-conserved, including in deep branching excavates such as Giardia, with several studies already implicating histone post-translational modifications in regulation of its pathogenesis and life cycle. However, further insights into Giardia chromatin dynamics have been hindered by a lack of site-specific knowledge of histone modifications. Using mass spectrometry, we have provided the first known molecular map of histone methylation, acetylation and phosphorylation modifications in Giardia core histones. We have identified over 50 previously unreported histone modifications including sites with established roles in epigenetic regulation, and co-occurring modifications indicative of post-translational modification crosstalk. These demonstrate conserved histone modifications in Giardia which are equivalent to many other eukaryotes, and suggest that similar epigenetic mechanisms are in place in this parasite. Further, we used sequence, domain and structural homology to annotate putative histone enzyme networks in Giardia, highlighting representative chromatin modifiers which appear sufficient for identified sites, particularly those from H3 and H4 variants. This study is to our knowledge the first and most comprehensive, complete and accurate view of Giardia histone post-translational modifications to date, and a substantial step towards understanding their associations in parasite development and virulence.     

Differential nuclease sensitivity of the ovalbumin and beta-globin chromatin regions in erythrocytes and oviduct cells of laying hen.

We have monitored the differential nuclease sensitivity of defined regions of the chicken genome in different cells using a method which combines restriction enzyme digestion and blotting to diazobenzyloxymethyl (DBM)-paper (see Ref. 11). By using different specific probes and by scanning the bands on the autoradiograms, it is possible to compare on the same blot the digestion patterns of similar-sized fragments from different regions of the genome corresponding to "active" and reference "inactive" genes. We have demonstrated the preferential sensitivity to DNaseI and micrococcal nuclease digestion of the ovalbumin gene region in hen oviduct chromatin. The beta-globin gene region (containing both an adult and an embryonic gene) is also preferentially digested by DNaseI in hen mature erythrocyte nuclei, but at a lower rate than the ovalbumin gene region in oviduct. These observations raise the possibility that there may be several types of preferential nuclease sensitivities, all characterized by increased rates of digestion but to different levels, the highest corresponding to the very actively transcribing genes.     

Comparison of nuclease digestion of polyoma virus nucleoprotein complex and mouse chromatin.

We digested polyoma virus nucleoprotein complex, isolated from disrupted virions, with micrococcal nuclease and DNase I. The results were compared with digestions of chromatin from mouse nuclei. The nucleosome "core" structures were similar, but the spacing of the nucleosomes in the isolated polymoma nucleoprotein complexes was irregular, whereas in mouse chromatin it was regular. The average nucleosome repeat length in each case was 190 to 200 base pairs. This figure suggests that, unless there are substantial stretches of free DNA, the polyoma nucleoprotein complex contains about 26 nucleosomes. The commonly used method of preparing the nucleoprotein complex by disruption of virions at pH 10.2 may lead to significant damage to the structure. Such damage may be more clearly revealed by the susceptibility of the DNA to nuclease digestion than by the usual criteria of sedimentation velocity and buoyant density.     

Digestion by micrococcal nuclease of mouse submandibular-salivary-gland chromatin.

Nucleic prepared from mouse submandibular salivary gland show marked fragility during isolation. Hwever, intact nuclei relatively free from cytoplasmic contamination were obtained by homogenization in buffers containing 0.88 M-sucrose, Ca2+, spermine, spermidine and the proteinase inhibitor aprotinin, followed by centrifugation through 2.2 M-sucrose. The kinetics of digestion by the micrococcal nuclease of chromatin in these nuclei are similar to those of chromatin from mouse liver nuclei. Base-pair size analysis of the solubilized DNA from both organs shows a stable high-molecular weight species of chromatin, which is further digested to mononucleosome and subnucleosome species. With extensive digestion the chromatin becomes insoluble. The mononucleosomes produced from salivary-gland chromatin after the inhibition of endogenous proteinase activity exhibit an s20,w value of 11S and contain histones H1, H2A, H2B, H3 and H4.     

Distribution of H1 histone in chromatin digested by micrococcal nuclease.

The relative amount of H1 histone associated with isolated nucleosomes from calf thymus was determined as a function of the extent of DNA digestion by micrococcal nuclease. Generally the amount of H1 histone associated with mononucleosomes decreases with increasing digestion until 60% of the original H1 remains associated with DNA 150 base pirs or less in size. Coincidentally, H1 histone increases relative to the other histones in aggregated material that sediments through sucrose gradients to form a pellet. However, the level of H1 histone remains at control values for oligonucleosomes (dimer to hexamer) over the 30% digestion range studied. An increase in ionic strength to 0.3 M NaCl in the density gradient reveals a different pattern of H1 binding, whereby the amount of H1 reflects the average size of the DNA fragments with which it is associated. Although there is significant binding to nucleosomes per se, it appears that the major ionic involvement of H1 is with internucleosomal spacer DNA.     

Noninteger pitch and nuclease sensitivity of chromatin DNA.

Assuming that variation of nuclease sensitivity along nucleosomal DNA can basically be attributed to orientations of sugar--phosphate bonds relative to histone core, the pitch of chromatin DNA is estimated to be 10.33--10.40 base pairs. This is in accordance both with the known measured average distance between cleavage sites (10.3--10.4 base pairs) and with published data on variation of relative sensitivities of these sites to nuclease attack. The variation can be explained solely as a result of the systematic change of orientation of sugar--phosphate bonds of sensitive sites without additional suggestions about local steric hindrances by histone molecules. According to the analysis locations of sites least sensitive to nuclease attack should not depend on kind of endonuclease though the stagger could differ. We conclude that the nucleosome core particle is axially symmetrical. The results strongly support the suggestion that DNA is wrapped around the histone octamer smoothly, without interruption of base-stacking interactions.     

Parental origin of chromatin in human monopronuclear zygotes revealed by asymmetric histone methylation patterns, differs between IVF and ICSI

In mouse zygotes, many post-translational histone modifications are asymmetrically present in male and female pronuclei. We investigated whether this principle could be used to determine the genetic composition of monopronuclear human zygotes in conventional IVF and ICSI. First we determined whether male female asymmetry is conserved from mouse to human by staining polypronuclear zygotes with antibodies against a subset of histone N-tail post-translational modifications. To analyze human monopronuclear zygotes, a modification, H3K9me3, was selected that is present in the maternal chromatin. After IVF a total of 45 monopronuclear zygotes were obtained. In 39 (87%) of zygotes a nonuniform staining pattern was observed, proof of a bi-parental origin and assumed to result into a diploid conception. Two zygotes showed no staining for the modification, indicating that the single pronucleus was of paternal origin. Four zygotes contained only maternally derived chromatin. ICSI-derived monopronuclear zygotes (n = 33) could also be divided into three groups based on the staining pattern of their chromatin: (1) of maternal origin (n = 15), (2) of paternal origin (n = 8) or (3) consisting of two chromatin domains as dominating in IVF (n = 10). Our data show that monopronuclear zygotes originating from IVF generally arise through fusion of parental chromatin after sperm penetration. Monopronuclear zygotes derived from ICSI in most cases contain uni-parental chromatin. The fact that chromatin was of paternal origin in 24% of ICSI and in 4% of the IVF zygotes confirms earlier results obtained by FISH on cleavage stages. Our findings are of clinical importance in IVF and ICSI practice.     

Synergistic and additive properties of the beta-globin locus control region (LCR) revealed by 5'HS3 deletion mutations: implication for LCR chromatin architecture

Deletion of the 234-bp core element of the DNase I hypersensitive site 3 (5'HS3) of the locus control region (LCR) in the context of a human beta-globin locus yeast artificial chromosome (beta-YAC) results in profound effects on globin gene expression in transgenic mice. In contrast, deletion of a 2.3-kb 5'HS3 region, which includes the 234-bp core sequence, has a much milder phenotype. Here we report the effects of these deletions on chromatin structure in the beta-globin locus of adult erythroblasts. The 234-bp 5'HS3 deletion abolished histone acetylation throughout the beta-globin locus; recruitment of RNA polymerase II (pol II) to the LCR and beta-globin gene promoter was reduced to a basal level; and formation of all the 5' DNase I hypersensitive sites of the LCR was disrupted. The 2.3-kb 5'HS3 deletion mildly reduced the level of histone acetylation but did not change the profile across the whole locus; the 5' DNase I hypersensitive sites of the LCR were formed, but to a lesser extent; and recruitment of pol II was reduced, but only marginally. These data support the hypothesis that the LCR forms a specific chromatin structure and acts as a single entity. Based on these results we elaborate on a model of LCR chromatin architecture which accommodates the distinct phenotypes of the 5'HS3 and HS3 core deletions.     

Deletions within the mouse beta-globin locus control region preferentially reduce beta(min) globin gene expression

The mouse beta-globin gene cluster is regulated, at least in part, by a locus control region (LCR) composed of several developmentally stable DNase I hypersensitive sites located upstream of the genes. In this report, we examine the level of expression of the beta(min) and beta(maj) genes in adult mice in which HS2, HS3, or HS5,6 has been either deleted or replaced by a selectable marker via homologous recombination in ES cells. Primer extension analysis of RNA extracted from circulating reticulocytes and HPLC analysis of globin chains from peripheral red blood cells revealed that all mutations that reduce the overall output of the locus preferentially decrease beta(min) expression over beta(maj). The implications of these findings for the mechanism by which the LCR controls expression of the beta(maj) and beta(min) promoters are discussed.     

A haplolethal locus uncovered by deletions in the mouse T complex

Proper levels of gene expression are important for normal mammalian development. Typically, altered gene dosage caused by karyotypic abnormalities results in embryonic lethality or birth defects. Segmental aneuploidy can be compatible with life but often results in contiguous gene syndromes. The ability to manipulate the mouse genome allows the systematic exploration of regions that are affected by alterations in gene dosage. To explore the effects of segmental haploidy in the mouse t complex on chromosome 17, radiation-induced deletion complexes centered at the Sod2 and D17Leh94 loci were generated in embryonic stem (ES) cells. A small interval was identified that, when hemizygous, caused specific embryonic lethal phenotypes (exencephaly and edema) in most fetuses. The penetrance of these phenotypes was background dependent. Additionally, evidence for parent-of-origin effects was observed. This genetic approach should be useful for identifying genes that are imprinted or whose dosage is critical for normal embryonic development.