Histone-modifying enzymes play a critical role in modulating chromatin dynamics. In this report we demonstrate that one of these enzymes, PR-Set7, and its corresponding histone modification, the monomethylation of histone H4 lysine 20 (H4K20), display a distinct cell cycle profile in mammalian cells: low at G 1 , increased during late S phase and G 2 , and maximal from prometaphase to anaphase. The lack of PR-Set7 and monomethylated H4K20 resulted in a number of aberrant phenotypes in several different mammalian cell types. These include the inability of cells to progress past G 2 , global chromosome condensation failure, aberrant centrosome amplification, and substantial DNA damage. By employing a catalytically dead dominant negative PR-Set7 mutant, we discovered that its mono-methyltransferase activity was required to prevent these phenotypes. Importantly, we demonstrate that all of the aberrant phenotypes associated with the loss of PR-Set7 enzymatic function occur independently of p53. Collectively, our findings demonstrate that PR-Set7 enzymatic activity is essential for mammalian cell cycle progression and for the maintenance of genomic stability, most likely by monomethylating histone H4K20. Our results predict that alterations of this pathway could result in gross chromosomal aberrations and aneuploidy.Dynamic alterations in chromatin structure are modulated, in part, by the post-translational modifications of the DNAassociated histone proteins. Specialized chromatin-modifying enzymes can phosphorylate, acetylate, ubiquitylate, or methylate specific amino acids within certain histones, and each of these modifications are associated with distinct biological events (1). One of the first histone modifications to be identified nearly forty-five years ago was the methylation of histone H4 lysine 20 (H4K20) 4 (2). Earlier biochemical studies linked H4K20 methylation to diverse biological events including transcriptional regulation, chromatin compaction, cell division, and the formation of heterochromatin (3-9). Importantly, it was also found that H4K20 is differentially methylated in vivo and therefore can be either mono-, di-, or trimethylated (10). Together, these findings strongly suggest that different methylated states of H4K20 may be involved in distinct biological processes, similar to what is observed for the various methylated states of histone H3 lysine 4 and 9 methylation (11, 12).Increasing evidence indicates that certain enzymes are responsible for the specific degree of histone lysine methylation (13). For example, the mono-and dimethylation of histone H3 lysine 9 in humans is mediated by the G9a enzyme, whereas trimethylation is mediated by the SUV39H1 enzyme (14,15). Similarly, the Suv4 -20 enzymes are responsible for di-and trimethylation in mammals (16,17). Trimethylated H4K20 is associated with repressed chromatin because it is targeted to constitutive heterochromatin, various repetitive elements, and imprinting control regions (16,18,19). Dimethylated H4K40 is more widely distributed within...

The specific post-translational modifications of the histone proteins are associated with specific DNA-templated processes, such as transcriptional activation or repression. To investigate the biological role(s) of histone H4 lysine 20 (H4 Lys-20) methylation, we created a novel panel of antibodies that specifically detected mono-, di-, or trimethylated H4 Lys-20. We report that the different methylated forms of H4 Lys-20 are compartmentalized within visually distinct, transcriptionally silent regions in the mammalian nucleus. Interestingly, direct comparison of methylated H4 Lys-20 with the different methylated states of histone H3 lysine 9 (H3 Lys-9) revealed significant overlap and exclusion between the specific groups of methyl modifications. Trimethylated H4 Lys-20 and H3 Lys-9 were both selectively enriched within pericentric heterochromatin. Similarly, monomethylated H4 Lys-20 and H3 Lys-9 partitioned together and the dimethylated forms partitioned together within the chromosome arms; however, the mono-and dimethylated modifications were virtually exclusive. These findings strongly suggest that the combinatorial presence or absence of the different methylated states of H4 Lys-20 and H3 Lys-9 define particular types of silent chromatin. Consistent with this, detailed analysis of monomethylated H4 Lys-20 and H3 Lys-9 revealed that both were preferentially and selectively enriched within the same nucleosome particle in vivo. Collectively, these findings define a novel trans-tail histone code involving monomethylated H4 Lys-20 and H3 Lys-9 that act cooperatively to mark distinct regions of silent chromatin within the mammalian epigenome.Within eukaryotic nuclei, DNA associates with nuclear proteins to form chromatin. The most fundamental structural unit of chromatin, the nucleosome, is composed of ϳ146 bp of DNA wrapped around an octamer of the core histone proteins H2A, H2B, H3, and H4 (1). Although much has been elucidated about chromatin in the last decade, it remains unclear how distinct functional domains of chromatin are established and maintained within living cells. It is well known that chromatin function is modulated, at least in part, by enzymes that posttranslationally modify specific amino acids on the histone proteins (2). Such post-translational modifications include acetylation, phosphorylation, ubiquitination, and methylation (3). Increasing evidence indicates that specific covalent modifications, alone or in combination, directly participate in specific downstream nuclear processes including transcription, replication, and repair (4). Thus, it is theorized that this "histone code" may serve to establish and maintain distinct functional domains that are epigenetically transmitted (5-7).One histone modification that has received increased attention is the methylation of lysine 20 on histone H4 (H4 Lys-20).2 Although this methylated histone lysine residue was identified over 40 years ago, the biological significance of this modification has remained enigmatic (8). Investigations into H4 Lys-20 ...

The most common chromosomal translocation in cancer, t(14;18), occurs at the bcl-2 major breakpoint region (Mbr) in follicular lymphomas. The 150-bp bcl-2 Mbr, which contains three breakage hotspots (peaks), has a single-stranded character and, hence, a non-B DNA conformation both in vivo and in vitro. Here, we use gel assays and electron microscopy to show that a triplexspecific antibody binds to the bcl-2 Mbr in vitro. Bisulfite reactivity shows that the non-B DNA structure is favored by, but not dependent upon, supercoiling and suggests a possible triplex conformation at one portion of the Mbr (peak I). We have used circular dichroism to test whether the predicted third strand of that suggested structure can indeed form a triplex with the duplex at peak I, and it does so with 1:1 stoichiometry. Using an intracellular minichromosomal assay, we show that the non-B DNA structure formation is critical for the breakage at the bcl-2 Mbr, because a 3-bp mutation that disrupts the putative peak I triplex also markedly reduces the recombination of the Mbr. A three-dimensional model of such a triplex is consistent with bond length, bond angle, and energetic restrictions (stacking and hydrogen bonding). We infer that an imperfect purine/purine/pyrimidine (R.R.Y) triplex likely forms at the bcl-2 Mbr in vitro, and in vivo recombination data favor this as the major DNA conformation in vivo as well.

Increasing evidence indicates that the post-translational modifications of the histone proteins play critical roles in all eukaryotic DNA-templated processes. To gain further biological insights into two of these modifications, the mono- and trimethylation of histone H4 lysine 20 (H4K20me1 and H4K20me3), ChIP-chip experiments were performed to identify the precise genomic regions on human chromosomes 21 and 22 occupied by these two modifications. Detailed analysis revealed that H4K20me1 was preferentially enriched within specific genes; most significantly between the first approximately 5% and 20% of gene bodies. In contrast, H4K20me3 was preferentially targeted to repetitive elements. Among genes enriched in H4K20me3, the modification was typically targeted to a small region approximately 1 kb upstream of transcription start. Our collective findings strongly suggest that H4K20me1 and H4K20me3 are both physically and functionally distinct. We next sought to determine the role of H4K20me1 in transcription since this has been controversial. Following the reduction of PR-Set7/Set8/KMT5a and H4K20me1 in cells by RNAi, all H4K20me1-associated genes analyzed displayed an approximately 2-fold increase in gene expression; H4K20me3-associated genes displayed no changes. Similar results were obtained using a catalytically dead dominant negative PR-Set7 indicating that H4K20me1, itself, is essential for the selective transcriptional repression of H4K20me1-associated genes. Furthermore, we determined that the H4K20me1-associated DNA sequences were sufficient to nucleate H4K20me1 and induce repression in vivo. Our findings reveal the molecular mechanisms of a mammalian transcriptional repressive pathway whereby the DNA sequences within specific gene bodies are sufficient to nucleate the monomethylation of H4K20 which, in turn, reduces gene expression by half.

We found that several major chromosomal fragile sites in human lymphomas, including the bcl-2 major breakpoint region, bcl-1 major translocation cluster, and c-Myc exon 1-intron 1 boundary, contain distinctive sequences of consecutive cytosines exhibiting a high degree of reactivity with the structure-specific chemical probe bisulfite. To assess the inherent structural variability of duplex DNA in these regions and to determine the range of structures reactive to bisulfite, we have performed bisulfite probing on genomic DNA in vitro and in situ; on duplex DNA in supercoiled and linearized plasmids; and on oligonucleotide DNA/DNA and DNA/2-O-methyl RNA duplexes. Bisulfite is significantly more reactive at the frayed ends of DNA duplexes, which is expected given that bisulfite is an established probe of single-stranded DNA. We observed that bisulfite also distinguishes between more subtle sequence/structural differences in duplex DNA. Supercoiled plasmids are more reactive than linear DNA; and sequences containing consecutive cytosines, namely GGGCCC, are more reactive than those with alternating guanine and cytosine, namely GCGCGC. Circular dichroism and x-ray crystallography show that the GGGCCC sequence forms an intermediate B/A structure. Molecular dynamics simulations also predict an intermediate B/A structure for this sequence, and probe calculations suggest greater bisulfite accessibility of cytosine bases in the intermediate B/A structure over canonical B-or A-form DNA. Electrostatic calculations reveal that consecutive cytosine bases create electropositive patches in the major groove, predicting enhanced localization of the bisulfite anion at homo-C tracts over alternating G/C sequences. These characteristics of homo-C tracts in duplex DNA may be associated with DNA-protein interactions in vivo that predispose certain genomic regions to chromosomal fragility.The sequence-specific structural variations of the dsDNA 5 helix are well documented (1-4). For example, DNA sequences with consecutive A (or T) bases can adopt a variation of the B-form helix with a narrower minor groove, whereas sequences with consecutive G (or C) bases are more prone to adopt the A-form helix under dehydrating conditions (5). The A-form helix observed in RNA and DNA-RNA hybrid duplexes is present during transcription and replication and sometimes when DNA is bound to proteins (6 -10). Most reported DNA crystal structures exhibit either the B-form or A-form helical structure, with a small fraction exhibiting an intermediate structure.A strong correlation exists between the propensity of a given DNA sequence to adopt a non-B-form structure and the interaction of solvent (water and ions) with the major and minor grooves (5), but neither the natural range of DNA conformations under physiologic solution-phase conditions nor how subtle variations in DNA structure may influence the activity of DNA binding proteins is well understood.The structure of a particular sequence of dsDNA is often inferred from its pattern of reactivity with struc...

The t(14;18) translocation involving the Ig heavy chain locus and the BCL-2 gene is the single most common chromosomal translocation in human cancer. Recently we reported in vitro and in vivo chemical probing data indicating that the 150-bp major breakpoint region (Mbr), which contains three breakage subregions (hotspots) (known as peaks I, II, and III), has single-stranded character and hence a non-B DNA conformation. Although we could document the non-B DNA structure formation at the bcl-2 Mbr, the structural studies were limited to chemical probing. Therefore, in the present study, we used multiple methods including circular dichroism to detect the non-B DNA at the bcl-2 Mbr. We established a new gel shift method to detect the altered structure at neutral pH on shorter DNA fragments containing the bcl-2 Mbr and analyzed the fine structural features. We found that the single-stranded region in the non-B DNA structure observed is stable for days and is asymmetric with respect to the Watson and Crick strands. It could be detected by oligomer probing, a bisulfite modification assay, or a P1 nuclease assay. We provide evidence that two different non-B conformations exist at peak I in addition to the single one observed at peak III. Finally we used mutagenesis and base analogue incorporation to show that the non-B DNA structure formation requires Hoogsteen pairing. These findings place major constraints on the location and nature of the non-B conformations assumed at peaks I and III of the bcl-2 Mbr.The t(14;18) translocation is the most common translocation in human cancer. It is the most important of all lymphoid cell translocations because follicular lymphomas account for nearly half of all non-Hodgkin's lymphomas. Analyses of the breakpoint regions have shown that the BCL-2 gene on chromosome 18 is joined to the J H segment of the immunoglobulin heavy chain locus on chromosome 14, while the reciprocal junction consists in fusion of a D H segment from the IgH locus on chromosome 14 with the remaining 3Ј BCL-2 untranslated region on chromosome 18 (1-9).The major breakpoint region (Mbr) 1 on chromosome 18 is in the 3Ј untranslated region of the BCL-2 gene (3, 5, 10 -13). The bcl-2 Mbr is distinctive among breakpoint regions (14). Nearly 80% of t(14;18) events occur in the 150-bp bcl-2 Mbr (11). The breakpoints are not at a specific nucleotide but rather are dispersed over a 150-bp region with relatively sharp boundaries (11,12,15). Within the 150 bp, there are three peaks of breakage, each about 15-20 bp in size (12). The characteristic precision and frequency profile within the bcl-2 Mbr strongly suggest some strong local regional specificity to the bcl-2 Mbr (14). We and others have previously provided experimental evidence that the RAG complex can mistakenly cleave at pseudosignals in lymphoid translocations involving LMO2, Ttg-1, and SIL (16 -19) but not at the bcl-2 Mbr (15, 18). Instead the 150-bp bcl-2 Mbr assumes a non-B form DNA structure within human cells at 20 -30% of alleles (15). The structure contai...

Histone modifications play important roles in all eukaryotic DNA‐templated processes. To gain further biological insights into two of these modifications, the mono‐ and trimethylation of histone H4 lysine 20 (H4K20me1 and H4K20me3), ChIP‐chip was performed to identify their precise locations on human chromosomes 21 and 22. Our findings indicate that H4K20me1 is preferentially enriched within specific genes whereas H4K20me3 is preferentially targeted to repetitive elements, strongly suggesting that H4K20me1 and H4K20me3 are physically and functionally distinct histone modifications. Consistent with this, depletion of H4K20me1 resulted in an ~2‐fold increase in the expression of H4K20me1‐associated genes while no changes in expression were observed in H4K20me3‐associated genes. In addition, we found that H4K20me1‐associated DNA sequences were sufficient to nucleate H4K20me1 and induce repression of a reporter gene in vivo. Our results reveal a new transcriptional regulatory pathway whereby the DNA sequences within specific gene bodies can establish H4K20me1 to induce their repression.