In senescent cells, specialized domains of transcriptionally silent senescence-associated heterochromatic foci (SAHF), containing heterochromatin proteins such as HP1, are thought to repress expression of proliferation-promoting genes. We have investigated the composition and mode of assembly of SAHF and its contribution to cell cycle exit. SAHF is enriched in a transcription-silencing histone H2A variant, macroH2A. As cells approach senescence, a known chromatin regulator, HIRA, enters PML nuclear bodies, where it transiently colocalizes with HP1 proteins, prior to incorporation of HP1 proteins into SAHF. A physical complex containing HIRA and another chromatin regulator, ASF1a, is rate limiting for formation of SAHF and onset of senescence, and ASF1a is required for formation of SAHF and efficient senescence-associated cell cycle exit. These data indicate that HIRA and ASF1a drive formation of macroH2A-containing SAHF and senescence-associated cell cycle exit, via a pathway that appears to depend on flux of heterochromatic proteins through PML bodies.

Human HIRA, ASF1a, ASF1b and CAF-1 are evolutionally conserved histone chaperones that form multiple functionally distinct chromatin assembly complexes, with roles linked to diverse nuclear process, such as DNA replication and formation of heterochromatin in senescent cells. We report the crystal structure of an ASF1a/HIRA heterodimer and a biochemical dissection of ASF1a's mutually exclusive interactions with HIRA and the p60 subunit of CAF-1. The HIRA B-domain forms an antiparallel β-hairpin that binds perpendicular to the strands of the β-sandwich of ASF1a, via β-sheet, salt-bridge and van der Waals contacts. The N-and C-terminal regions of ASF1a and ASF1b determine the different affinities of these two proteins for HIRA, by contacting regions outside the HIRA B-domain. CAF-1 p60 also employs B-domain-like motifs for binding to ASF1a, thereby competing with HIRA. Together, these studies begin to define the molecular determinants of assembly of functionally diverse macromolecular histone chaperone complexes. KeywordsHistone Deposition; Chromatin Regulation; Histone Chaperones; ASF1; HIRA; CAF-1The basic repeating unit of eukaryotic chromatin is the core nucleosome. Each core nucleosome consists of a histone (H3/H4) 2 heterotetramer, two histone (H2A/H2B) heterodimers and about 147 base pairs of DNA. During chromatin assembly, histone chaperones donate histones to DNA 1 . In human cells, the deposition of the histone H3/H4 complex involves four histone chaperones, Chromatin Assembly Factor 1 (CAF-1), HIstone Regulatory Homolog A (HIRA), and two Anti-Silencing Factor paralogs (ASF1a and ASF1b).CAF-1, an evolutionarily conserved heterotrimeric chaperone comprised of p150, p60 and p48 subunits, mediates DNA synthesis-coupled chromatin assembly in S-phase 1,2 . CAF-1 is also 4 Correspondence should be addressed to R.M. or P.D. A., Ronen Marmorstein, Tel: (215) FAX: (215) marmor@wistar.org, Peter D. Adams, Tel: (215) FAX: (215) 728 3616, Peter.Adams@fccc.edu. 5 Y.T. and M.V.P contributed equally to this work.The coordinates of the ASF1a/HIRA complex structure has been deposited with the Protein Data Bank (PDB) with access code 2I32. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. AUTHOR CONTRIBUTIONSY.T. and M.V.P. designed and carried out experiments reported in the manuscript, and prepared manuscript figures and text; K.Z., M.G., A.C. and R.D. carried out preliminary studies that led to experiments reported in the manuscript; P.D.A and R.M. designed and supervised experiments and prepared manuscript text. All authors read and approved the submitted manuscript. NIH Public Access Author ManuscriptNat Struct Mol Biol. Author manuscript; available in PMC 2010 September 6. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript required for chromatin assembly coupled to DNA repair 3-8 , formation of specialized chromatin structures, such as transcriptionally silent chromatin at telomeres, rDNA yeast mating loci, and plant gene loci 3,...

SUMMARY IκB kinase α (IKKα) activity is required for ErbB2-induced mammary tumorigenesis. Here, we show that IKKα and its activator, NF-κB-inducing kinase (NIK), support the expansion of tumor-initiating cells (TICs) that copurify with a CD24medCD49fhi population from premalignant ErbB2-expressing mammary glands. Upon activation, IKKα enters the nucleus, phosphorylates the cyclin-dependent kinase (CDK) inhibitor p27/Kip1, and stimulates its nuclear export or exclusion. Reduced p27 expression rescues mammary tumorigenesis in mice deficient in IKKα kinase activity and restores TIC self-renewal. IKKα is also likely to be involved in human breast cancer, where its expression shows an inverse correlation with metastasis-free survival, and its presence in the nucleus of invasive ductal carcinomas (IDCs) is associated with decreased nuclear p27 abundance.

Mice bearing a v-Myc myelocytomatosis viral oncogene homolog ( c-Myc ) transgene controlled by an Ig-alpha heavy-chain enhancer (i Myc Cα mice) rarely develop lymphomas but instead have increased rates of memory B-cell turnover and impaired antibody responses to antigen. We found that male progeny of i Myc Cα mice mated with mice transgenic (Tg) for CD257 (B-cell activating factor, BAFF) developed CD5 + B-cell leukemia resembling human chronic lymphocytic leukemia (CLL), which also displays a male gender bias. Surprisingly, leukemic cells of Myc/Baff Tg mice expressed higher levels of c-Myc than did B cells of i Myc Cα mice. We found that CLL cells of many patients with progressive disease also expressed high amounts of c-MYC, particularly CLL cells whose survival depends on nurse-like cells (NLC), which express high-levels of BAFF. We find that BAFF could enhance CLL-cell expression of c-MYC via activation the canonical IκB kinase (IKK)/NF-κB pathway. Inhibition of the IKK/NF-κB pathway in mouse or human leukemia cells blocked the capacity of BAFF to induce c-MYC or promote leukemia-cell survival and significantly impaired disease progression in Myc/Baff Tg mice. This study reveals an important relationship between BAFF and c-MYC in CLL which may affect disease development and progression, and suggests that inhibitors of the canonical NF-κB pathway may be effective in treatment of patients with this disease.

Recent studies indicate that human pluripotent stem cell (PSC)-based therapies hold great promise in Parkinson's disease (PD). Clinical studies have shown that grafted fetal neural tissue can achieve considerable biochemical and clinical improvements in PD. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells offer a good alternative because they are derived from unfertilized oocytes without destroying viable human embryos and can be used to generate an unlimited supply of neural stem cells for transplantation. Here we evaluate for the first time the safety and engraftment of human parthenogenetic stem cell-derived neural stem cells (hpNSCs) in two animal models: 6-hydroxydopamine (6-OHDA)-lesioned rodents and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated nonhuman primates (NHPs). In both rodents and nonhuman primates, we observed successful engraftment and higher dopamine levels in hpNSC-transplanted animals compared to vehicle control animals, without any adverse events. These results indicate that hpNSCs are safe, well tolerated, and could potentially be a source for cell-based therapies in PD.

Cell therapy has attracted considerable interest as a promising therapeutic alternative for patients with Parkinson's disease (PD). Clinical studies have shown that grafted fetal neural tissue can achieve considerable biochemical and clinical improvements in PD. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells offer a good alternative because they are derived from unfertilized oocytes without destroying potentially viable human embryos and can be used to generate an unlimited supply of neural cells for transplantation. We have previously reported that human parthenogenetic stem cellderived neural stem cells (hpNSCs) successfully engraft, survive long term, and increase brain dopamine (DA) levels in rodent and nonhuman primate models of PD. Here we report the results of a 12-month transplantation study of hpNSCs in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned African green monkeys with moderate to severe clinical parkinsonian symptoms. The hpNSCs manufactured under current good manufacturing practice (cGMP) conditions were injected bilaterally into the striatum and substantia nigra of immuno suppressed monkeys. Transplantation of hpNSCs was safe and well tolerated by the animals with no dyskinesia, tumors, ectopic tissue formation, or other test article-related serious adverse events. We observed that hpNSCs promoted behavioral recovery; increased striatal DA concentration, fiber innervation, and number of dopaminergic neurons; and induced the expression of genes and pathways downregulated in PD compared to vehicle control animals. These results provide further evidence for the clinical translation of hpNSCs and support the approval of the world's first pluripotent stem cell-based phase I/IIa study for the treatment of PD (Clinical Trial Identifier NCT02452723).

Human pluripotent stem cells (PSC) have the potential to revolutionize regenerative medicine. However undifferentiated PSC can form tumors and strict quality control measures and safety studies must be conducted before clinical translation. Here we describe preclinical tumorigenicity and biodistribution safety studies that were required by the US Food and Drug Administration (FDA) and Australian Therapeutic Goods Administration (TGA) prior to conducting a Phase I clinical trial evaluating the safety and tolerability of human parthenogenetic stem cell derived neural stem cells ISC-hpNSC for treating Parkinson’s disease (ClinicalTrials.gov Identifier NCT02452723). To mitigate the risk of having residual PSC in the final ISC-hpNSC population, we conducted sensitive in vitro assays using flow cytometry and qRT-PCR analyses and in vivo assays to determine acute toxicity, tumorigenicity and biodistribution. The results from these safety studies show the lack of residual undifferentiated PSC, negligible tumorigenic potential by ISC-hpNSC and provide additional assurance to their clinical application.

Varicella-zoster virus (VZV) immediate-early 63 protein (IE63) is abundantly expressed during both acute infection in vitro and latent infection in human ganglia.Using the yeast two-hybrid system, we found that VZV IE63 interacts with human antisilencing function 1 protein (ASF1). ASF1 is a nucleosome assembly factor which is a member of the H3/H4 family of histone chaperones. IE63 coimmunoprecipitated and colocalized with ASF1 in transfected cells expressing IE63 and in VZV-infected cells. IE63 also colocalized with ASF1 in both lytic and latently VZV-infected enteric neurons. ASF1 exists in two isoforms, ASF1a and ASF1b, in mammalian cells. IE63 preferentially bound to ASF1a, and the amino-terminal 30 amino acids of ASF1a were critical for its interaction with IE63. VZV IE63 amino acids 171 to 208 and putative phosphorylation sites of IE63, both of which are critical for virus replication and latency in rodents, were important for the interaction of IE63 with ASF1. Finally, we found that IE63 increased the binding of ASF1 to histone H3.1 and H3.3, which suggests that IE63 may help to regulate levels of histones in virus-infected cells. Since ASF1 mediates eviction and deposition of histones during transcription, the interaction of VZV IE63 with ASF1 may help to regulate transcription of viral or cellular genes during lytic and/or latent infection.Varicella-zoster virus (VZV) is a neurotrophic human alphaherpesvirus. Primary infection causes chicken pox, or varicella, which results in a lifelong latent infection in trigeminal and dorsal root ganglia (30,32,35). Later in life, as a result of waning immune status due to aging or immunosuppression, VZV reactivates, resulting in shingles, or herpes zoster.During latency, VZV expresses at least six different viral transcripts (11,12,25,40). Open reading frame 63 (ORF63) is the most abundant VZV transcript expressed during latency (11). VZV ORF63 encodes a 278-amino-acid protein with immediate-early (IE) expression kinetics, referred to as IE63 (13). IE63 has been detected in latently infected human (18,25,36,38) and experimentally infected rodent (13, 26) ganglia. While IE63 is predominantly expressed in the nucleus during lytic replication in vitro, during latency the protein is detected in the cytoplasm of sensory neurons (18,36,38).IE63 is also abundantly expressed during lytic VZV replication (13, 28, 54). In VZV-infected cells, IE63 is extensively phosphorylated by VZV ORF47 protein kinase (27) and by cellular casein kinases (5, 54). IE63 is a component of the VZV tegument (28) and represses the activity of a number of VZV and heterologous viral and cellular promoters (5,14). IE63 is required to overcome the host innate response mediated by alpha interferon (3) and to inhibit apoptosis in primary human neuronal cells infected with VZV in culture (24). IE63 binds to RNA polymerase II and VZV IE62, the major viral transactivator, and enhances the activity of the VZV gI promoter (37).In this study, we show that VZV IE63 interacts with human antisilencing function ...