Jayanth V. Chodaparambil

The effect of H3K79 dimethylation and H4K20 trimethylation on nucleosome and chromatin structure.

Histone methylation regulates chromatin function dependent on the site and degree of the modification. In addition to creating binding sites for proteins, methylated lysine residues are likely to influence chromatin structure directly. Here we present crystal structures of nucleosomes reconstituted with methylated histones and investigate the folding behavior of resulting arrays. We demonstrate that dimethylation of histone H3 at lysine residue 79 locally alters the nucleosomal surface, whereas trimethylation of H4 at lysine residue 20 affects higher-order structure.

A charged and contoured surface on the nucleosome regulates chromatin compaction.

Local nucleosome-nucleosome interactions in cis drive chromatin folding, whereas interactions in trans lead to fiber-fiber oligomerization. Here we show that peptides derived from the histone H4 tail and Kaposis sarcoma herpesvirus LANA protein can replace the endogenous H4 tail, resulting in array folding and oligomerization. Neutralization of a LANA binding site on the histone surface enhanced rather than abolished nucleosome-nucleosome interactions. We maintain that the contoured nucleosome surface is centrally involved in regulating chromatin condensation.

Structure and dynamic properties of nucleosome core particles

It is now widely recognized that the packaging of genomic DNA, together with core histones, linker histones, and other functional proteins into chromatin profoundly influences nuclear processes such as transcription, replication, DNA repair, and recombination. Whereas earlier structural studies portrayed nucleosomes (the basic repeating unit of chromatin) as monolithic and static macromolecular assemblies, we now know that they are highly dynamic and capable of extensive crosstalk with the cellular machinery. Histone variants have evolved to locally alter chromatin structure, whereas histone chaperones and other cellular factors promote histone exchange and chromatin fluidity. Both of these phenomena likely facilitate interconversion between different chromatin states that show varying degrees of transcriptional activity.

Kaposi’s Sarcoma-Associated Herpesvirus LANA Hitches a Ride on the Chromosome

Kaposi’s sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and has an etiologic role in Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. Survival in rapidly dividing cells depends on a carefully orchestrated chain of events. The viral genome, or episome, must replicate in concert with cellular genetic material, and then efficiently segregate to progeny nuclei. KSHV achieves this through its latency associated nuclear antigen (LANA), which simultaneously binds to viral DNA and mitotic chromosomes to efficiently partition episomes. LANA’s N-terminal region has been shown to be essential for efficient KSHV DNA replication and tethering to mitotic chromosomes. The precise mechanism by which LANA attaches to host chromosomes has been an area of active investigation. We recently reported that this association is mediated by the chromatin components histones H2A and H2B. Binding between LANA and these proteins was demonstrated in vivo and in vitro, and use of an H2A-H2B depleted system demonstrated their central role in LANA’s chromosome binding. Further, we provided a structural description of the interaction of LANA’s N-terminal chromosome association region with the nucleosome using x-ray crystallography. Our data offer further insight into the mechanism of KSHV latency, and also reveal a new concept for a role of the nucleosome as a docking site for other proteins.

Molecular functions of the TLE tetramerization domain in Wnt target gene repression

Wnt signaling activates target genes by promoting association of the co‐activator β‐catenin with TCF/LEF transcription factors. In the absence of β‐catenin, target genes are silenced by TCF‐mediated recruitment of TLE/Groucho proteins, but the molecular basis for TLE/TCF‐dependent repression is unclear. We describe the unusual three‐dimensional structure of the N‐terminal Q domain of TLE1 that mediates tetramerization and binds to TCFs. We find that differences in repression potential of TCF/LEFs correlates with their affinities for TLE‐Q, rather than direct competition between β‐catenin and TLE for TCFs as part of an activation–repression switch. Structure‐based mutation of the TLE tetramer interface shows that dimers cannot mediate repression, even though they bind to TCFs with the same affinity as tetramers. Furthermore, the TLE Q tetramer, not the dimer, binds to chromatin, specifically to K20 methylated histone H4 tails, suggesting that the TCF/TLE tetramer complex promotes structural transitions of chromatin to mediate repression.

Nucleosome structure and function.

It is now widely recognized that the packaging of genomic DNA, together with core histones, linker histones, and other functional proteins into chromatin profoundly influences nuclear processes such as transcription, replication, DNA repair, and recombination. How chromatin structure modulates the expression of knowledge encoded in eukaryotic genomes, and how these processes take place within the context of a highly complex and compacted genomic chromatin environment remains a major unresolved question in biology. Here we review recent advances in nucleosome structure and dynamics.

Reactive Oxygen Species Regulate Nucleostemin Oligomerization and Protein Degradation

Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H2O2) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H2O2 impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-l-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation.