Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder caused by mutations in the cohesin-loading protein NIPBL1,2 for nearly 60% of individuals with classical CdLS3-5 and in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands6,7. In humans, the multi-subunit complex cohesin is comprised of SMC1, SMC3, RAD21 and a STAG protein to form a ring structure proposed to encircle sister chromatids to mediate sister chromatid cohesion (SCC)8 as well as play key roles in gene regulation9. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin10-13 and in yeast, HOS1, a class I histone deacetylase, deacetylates SMC3 during anaphase14-16. Here we report the identification of HDAC8 as the vertebrate SMC3 deacetylase as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation (SMC3-ac) and inefficient dissolution of the “used” cohesin complex released from chromatin in both prophase and anaphase. While SMC3 with retained acetylation is loaded onto chromatin, ChIP-Seq analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.

dLimitless reproductive potential is one of the hallmarks of cancer cells. This ability is due to the maintenance of telomeres, erosion of which causes cellular senescence or death. While most cancer cells activate telomerase, a telomere-elongating enzyme, it remains elusive as to why cancer cells often maintain shorter telomeres than the cells in the surrounding normal tissues. Here, we show that forced telomere elongation in cancer cells promotes their differentiation in vivo. We elongated the telomeres of human prostate cancer cells that possess short telomeres by enhancing their telomerase activity. The resulting cells had long telomeres and retained the ability to form tumors in nude mice. Strikingly, these tumors exhibited many duct-like structures and reduced N-cadherin expression, reminiscent of well-differentiated adenocarcinoma. These changes were caused by telomere elongation and not by enhanced telomerase activity. Gene expression profiling revealed that tumor formation was accompanied by the expression of innate immune system-related genes, which have been implicated in maintaining tumor cells in an undifferentiated state and poor-prognosis cancers. In tumors derived from the telomere-elongated cells, upregulation of such gene sets is not observed. Our observations suggest a functional contribution of short telomeres to tumor malignancy by regulation of cancer cell differentiation.

Werner syndrome (WS) is a premature aging disorder characterized by chromosomal instability and cancer predisposition. Mutations in WRN are responsible for the disease and cause telomere dysfunction, resulting in accelerated aging. Recent studies have revealed that cells from WS patients can be successfully reprogrammed into induced pluripotent stem cells (iPSCs). In the present study, we describe the effects of long-term culture on WS iPSCs, which acquired and maintained infinite proliferative potential for self-renewal over 2 years. After long-term cultures, WS iPSCs exhibited stable undifferentiated states and differentiation capacity, and premature upregulation of senescence-associated genes in WS cells was completely suppressed in WS iPSCs despite WRN deficiency. WS iPSCs also showed recapitulation of the phenotypes during differentiation. Furthermore, karyotype analysis indicated that WS iPSCs were stable, and half of the descendant clones had chromosomal profiles that were similar to those of parental cells. These unexpected properties might be achieved by induced expression of endogenous telomerase gene during reprogramming, which trigger telomerase reactivation leading to suppression of both replicative senescence and telomere dysfunction in WS cells. These findings demonstrated that reprogramming suppressed premature senescence phenotypes in WS cells and WS iPSCs could lead to chromosomal stability over the long term. WS iPSCs will provide opportunities to identify affected lineages in WS and to develop a new strategy for the treatment of WS.

Telomere erosion causes cell mortality, suggesting that longer telomeres enable more cell divisions. In telomerase-positive human cancer cells, however, telomeres are often kept shorter than those of surrounding normal tissues. Recently, we showed that cancer cell telomere elongation represses innate immune genes and promotes their differentiation in vivo. This implies that short telomeres contribute to cancer malignancy, but it is unclear how such genetic repression is caused by elongated telomeres. Here, we report that telomeric repeat-containing RNA (TERRA) induces a genome-wide alteration of gene expression in telomere-elongated cancer cells. Using three different cell lines, we found that telomere elongation up-regulates TERRA signal and down-regulates innate immune genes such as STAT1, ISG15 and OAS3 in vivo. Ectopic TERRA oligonucleotides repressed these genes even in cells with short telomeres under three-dimensional culture conditions. This appeared to occur from the action of G-quadruplexes (G4) in TERRA, because control oligonucleotides had no effect and a nontelomeric G4-forming oligonucleotide phenocopied the TERRA oligonucleotide. Telomere elongation and G4-forming oligonucleotides showed similar gene expression signatures. Most of the commonly suppressed genes were involved in the innate immune system and were up-regulated in various cancers. We propose that TERRA G4 counteracts cancer malignancy by suppressing innate immune genes.

Tobacco mosaic virus (TMV) encodes a 30-kDa movement protein (MP) which enables viral movement from cell to cell. It is, however, unclear whether the 126-and 183-kDa replicase proteins are involved in the cell-to-cell movement of TMV. In the course of our studies into TMV-R, a strain with a host range different from that of TMV-U1, we have obtained an interesting chimeric virus, UR-hel. The amino acid sequence differences between UR-hel and TMV-U1 are located only in the helicase-like domain of the replicase. Interestingly, UR-hel has a defect in its cell-to-cell movement. The replication of UR-hel showed a level of replication of the genome, synthesis, and accumulation of MP similar to that observed in TMV-U1-inoculated protoplasts. Such observations support the hypothesis that the replicase coding region may in some fashion be involved in cell-to-cell movement of TMV.Tobacco mosaic virus (TMV) has a positive-sense singlestranded RNA genome that encodes four proteins (7). The 126-and 183-kDa replicases are translated directly from the genomic RNA using the same first initiation codon. The movement protein (MP) and the coat protein (CP) are translated from their respective subgenomic mRNAs, which are synthesized during the replication cycle. Functions have been assigned to the genes mainly according to the phenotypic lesions of deletion or substitution mutants of each protein. It has been shown that the 126-and 183-kDa replicases are involved in intracellular replication (12), that the MP is involved in cellto-cell movement (5, 18), and that the CP is involved in longdistance movement (9, 19). TMV requires MP but not CP for cell-to-cell movement. It is as yet unclear whether or not replicase directly takes part in cell-to-cell movement, since cellto-cell movement prior to replication has not been observed.TMV-R, the rakkyo strain, which exhibits distinct host range differences from the common strain of TMV-U1, was recently reported (14). TMV-R infects rakkyo (Allium chinense), a monocot host that TMV-U1 is unable to infect. TMV-R causes only latent infection of Nicotiana tabacum cv. Bright Yellow (BY) in inoculated leaves, whereas TMV-U1 infects BY tobacco plants systemically and induces mosaic virus symptoms (14). The overall sequence homologies between TMV-U1 and TMV-R are 94% at the nucleotide level and 96 to 98% at the amino acid sequence level of the encoded proteins (3). Chen et al. constructed a series of chimeric viruses between the two strains (4). When chimeric viruses in which the replicase domain was derived from TMV-U1 were used, mosaic virus symptoms were observed on BY tobacco plants. In contrast, infection using chimeric viruses in which the replicase proteins had been derived from TMV-R resulted in only latent infection in BY tobacco plants with inoculated leaves. The phenotypes of all the chimeric viruses on BY tobacco plants were