LSD2/KDM1B and its cofactor NPAC/GLYR1 endow a structural and molecular model for regulation of H3K4 demethylation.

Dynamic regulation of histone methylation represents a fundamental epigenetic mechanism underlying eukaryotic gene regulation, yet little is known about how the catalytic activities of histone demethylases are regulated. Here, we identify and characterize NPAC/GLYR1 as an LSD2/KDM1b-specific cofactor that stimulates H3K4me1 and H3K4me2 demethylation. We determine the crystal structures of LSD2 alone and LSD2 in complex with the NPAC linker region in the absence or presence of histone H3 peptide, at resolutions of 2.9, 2.0, and 2.25 Å, respectively. These crystal structures and further biochemical characterization define a dodecapeptide of NPAC (residues 214-225) as the minimal functional unit for its cofactor activity and provide structural determinants and a molecular mechanism underlying the intrinsic cofactor activity of NPAC in stimulating LSD2-catalyzed H3K4 demethylation. Thus, these findings establish a model for how a cofactor directly regulates histone demethylation and will have a significant impact on our understanding of catalytic-activity-based epigenetic regulation.

Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells.

DNA methylation at the 5 position of cytosine (5mC) in the mammalian genome is a key epigenetic event critical for various cellular processes. The ten-eleven translocation (Tet) family of 5mC-hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), offers a way for dynamic regulation of DNA methylation. Here we report that Tet1 binds to unmodified C or 5mC- or 5hmC-modified CpG-rich DNA through its CXXC domain. Genome-wide mapping of Tet1 and 5hmC reveals mechanisms by which Tet1 controls 5hmC and 5mC levels in mouse embryonic stem cells (mESCs). We also uncover a comprehensive gene network influenced by Tet1. Collectively, our data suggest that Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting 5mC to 5hmC through hydroxylase activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 targets, ultimately contributing to mESC differentiation and the onset of embryonic development.

Human LSD2/KDM1b/AOF1 regulates gene transcription by modulating intragenic H3K4me2 methylation.

Dynamic histone H3K4 methylation is an important epigenetic component of transcriptional regulation. However, most of our current understanding of this histone mark is confined to the regulation of transcriptional initiation. We now show that human LSD2/KDM1b/AOF1, the human homolog of LSD1, is an H3K4me1/2 demethylase that specifically regulates histone H3K4 methylation within intragenic regions of its target genes. Genome-wide mapping reveals that LSD2 associates predominantly with the gene bodies of actively transcribed genes, but is markedly absent from promoters. Depletion of endogenous LSD2 results in an increase of H3K4me2 as well as a decrease of H3K9me2 at LSD2-binding sites and a consequent dysregulation of target gene transcription. Furthermore, characterization of the LSD2 complex reveals that LSD2 forms active complexes with euchromatic histone methyltransferases G9a and NSD3 as well as cellular factors involved in transcription elongation. These data provide a possible molecular mechanism linking LSD2 to transcriptional regulation after initiation.

Role of Kaposi's sarcoma-associated herpesvirus C-terminal LANA chromosome binding in episome persistence.

Kaposi's sarcoma-associated herpesvirus (KSHV) LANA is an 1,162-amino-acid protein that tethers terminal repeat (TR) DNA to mitotic chromosomes to mediate episome persistence in dividing cells. C-terminal LANA self-associates to bind TR DNA. LANA contains independent N- and C-terminal chromosome binding regions. N-terminal LANA binds histones H2A/H2B to attach to chromosomes, and this binding is essential for episome persistence. We now investigate the role of C-terminal chromosome binding in LANA function. Alanine substitutions for LANA residues (1068)LKK(1070) and (1125)SHP(1127) severely impaired chromosome binding but did not reduce the other C-terminal LANA functions of self-association or DNA binding. The (1068)LKK(1070) and (1125)SHP(1127) substitutions did not reduce LANA's inhibition of RB1-induced growth arrest, transactivation of the CDK2 promoter, or C-terminal LANA's inhibition of p53 activation of the BAX promoter. When N-terminal LANA was wild type, the (1068)LKK(1070) and (1125)SHP(1127) substitutions also did not reduce LANA chromosome association or episome persistence. However, when N-terminal LANA binding to chromosomes was modestly diminished, the substitutions in (1068)LKK(1070) and (1125)SHP(1127) dramatically reduced both LANA chromosome association and episome persistence. These data suggest a model in which N- and C-terminal LANA cooperatively associates with chromosomes to mediate full-length LANA chromosome binding and viral persistence.

The KSHV latency-associated nuclear antigen: a multifunctional protein.

Kaposi's sarcoma(KS)-associated herpes virus (KSHV) or human herpesvirus 8 (HHV-8) is highly associated with KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease, an aggressive lymphoproliferative disorder (1-3). Most tumor cells are latently infected with KSHV in which a small subset of viral genes are expressed (4-6). Of these latently expressed genes, the latency-associated nuclear antigen (LANA1, LNA, or LNA1) is the only protein consistently shown to be highly expressed by in situ hybridization and immunohistochemistry (7-10). In the past few years multiple functions have been demonstrated for LANA1. Here we review LANA1's roles in KSHV infection. Topics discussed include LANA1's roles in episome persistence, regulation of transcription and interaction with cellular proteins.

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 Kaposi's 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.

Determination of Kaposi's sarcoma-associated herpesvirus C-terminal latency-associated nuclear antigen residues mediating chromosome association and DNA binding.

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen (LANA) tethers viral terminal repeat (TR) DNA to mitotic chromosomes to mediate episome persistence. The 1,162-amino-acid LANA protein contains both N- and C-terminal chromosome attachment regions. The LANA C-terminal domain self-associates to specifically bind TR DNA and mitotic chromosomes. Here, we used alanine scanning substitutions spanning residues 1023 to 1145 to investigate LANA self-association, DNA binding, and C-terminal chromosome association. No residues were essential for LANA oligomerization, as assayed by coimmunoprecipitation experiments, consistent with redundant roles for amino acids in self-association. Different subsets of amino acids were important for DNA binding, as assayed by electrophoretic mobility shift assay, and mitotic chromosome association, indicating that distinct C-terminal LANA subdomains effect DNA and chromosome binding. The DNA binding domains of LANA and EBNA1 are predicted to be structurally homologous; certain LANA residues important for DNA binding correspond to those with roles in EBNA1 DNA binding, providing genetic support for at least partial structural homology. In contrast to the essential role of N-terminal LANA chromosome targeting residues in DNA replication, deficient C-terminal chromosome association did not reduce LANA-mediated DNA replication.

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.

The nucleosomal surface as a docking station for Kaposi's sarcoma herpesvirus LANA.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) mediates viral genome attachment to mitotic chromosomes. We find that N-terminal LANA docks onto chromosomes by binding nucleosomes through the folded region of histones H2A-H2B. The same LANA residues were required for both H2A-H2B binding and chromosome association. Further, LANA did not bind Xenopus sperm chromatin, which is deficient in H2A-H2B; chromatin binding was rescued after assembly of nucleosomes containing H2A-H2B. We also describe the 2.9-angstrom crystal structure of a nucleosome complexed with the first 23 LANA amino acids. The LANA peptide forms a hairpin that interacts exclusively with an acidic H2A-H2B region that is implicated in the formation of higher order chromatin structure. Our findings present a paradigm for how nucleosomes may serve as binding platforms for viral and cellular proteins and reveal a previously unknown mechanism for KSHV latency.

The Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 N terminus is essential for chromosome association, DNA replication, and episome persistence.

To persist in latently infected, proliferating cells, Kaposi's sarcoma-associated herpesvirus (KSHV) episomes must replicate and efficiently segregate to progeny nuclei. Episome persistence in uninfected cells requires latency-associated nuclear antigen 1 (LANA1) in trans and cis-acting KSHV terminal repeat (TR) DNA. The LANA1 C terminus binds TR DNA, and LANA1 mediates TR-associated DNA replication in transient assays. LANA1 also concentrates at sites of KSHV TR DNA episomes along mitotic chromosomes, consistent with a tethering role to efficiently segregate episomes to progeny nuclei. LANA1 amino acids 5 to 22 constitute a chromosome association region (Piolot et al., J. Virol. 75:3948-3959, 2001). We now investigate LANA1 residues 5 to 22 with scanning alanine substitutions. Mutations targeting LANA1 5GMR7, 8LRS10, and 11GRS13 eliminated chromosome association, DNA replication, and episome persistence. LANA1 mutated at 14TG15 retained the ability to associate with chromosomes but was partially deficient in DNA replication and episome persistence. These results provide genetic support for a key role of the LANA1 N terminus in chromosome association, LANA1-mediated DNA replication, and episome persistence.

KSHV LANA1 binds DNA as an oligomer and residues N-terminal to the oligomerization domain are essential for DNA binding, replication, and episome persistence.

Latency-associated nuclear antigen 1 (LANA1) binds to Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeat (TR) DNA to mediate episome replication and persistence. LANA1 concentrates at sites of TR DNA along mitotic chromosomes, consistent with tethering KSHV DNA to chromosomes for efficient segregation of episomes to progeny nuclei. We now investigate LANA1 C-terminus self-association and DNA binding. The TR DNA binding domain was localized to LANA1 residues 996-1139. Scanning deletions within this region ablated both LANA1 oligomerization and DNA binding, consistent with a requirement for oligomerization to bind DNA. Furthermore, LANA1 bound TR DNA as an oligomer. Deletion of amino acids 1007-1021, N-terminal to the LANA1 oligomerization domain, ablated DNA binding, DNA replication, and episome persistence, implicating these residues in contacting DNA. Indeed, LANA1 residues 1007-1021 correspond to EBNA1 residues that contact the cognate sequence. Like EBNA1, the LANA1 DNA-binding domain has oligomerization activity and critical residues essential for recognizing DNA.