Structural transitions of centromeric chromatin regulate the cell cycle-dependent recruitment of CENP-N.

Specific recognition of centromere-specific histone variant CENP-A-containing chromatin by CENP-N is an essential process in the assembly of the kinetochore complex at centromeres prior to mammalian cell division. However, the mechanisms of CENP-N recruitment to centromeres/kinetochores remain unknown. Here, we show that a CENP-A-specific RG loop (Arg80/Gly81) plays an essential and dual regulatory role in this process. The RG loop assists the formation of a compact "ladder-like" structure of CENP-A chromatin, concealing the loop and thus impairing its role in recruiting CENP-N. Upon G1/S-phase transition, however, centromeric chromatin switches from the compact to an open state, enabling the now exposed RG loop to recruit CENP-N prior to cell division. Our results provide the first insights into the mechanisms by which the recruitment of CENP-N is regulated by the structural transitions between compaction and relaxation of centromeric chromatin during the cell cycle.

Histone variant H2A.Z regulates nucleosome unwrapping and CTCF binding in mouse ES cells.

Nucleosome is the basic structural unit of chromatin, and its dynamics plays critical roles in the regulation of genome functions. However, how the nucleosome structure is regulated by histone variants in vivo is still largely uncharacterized. Here, by employing Micrococcal nuclease (MNase) digestion of crosslinked chromatin followed by chromatin immunoprecipitation (ChIP) and paired-end sequencing (MNase-X-ChIP-seq), we mapped unwrapping states of nucleosomes containing histone variant H2A.Z in mouse embryonic stem (ES) cells. We found that H2A.Z nucleosomes are more enriched with unwrapping states compared with canonical nucleosomes. Interestingly, +1 H2A.Z nucleosomes with 30-80 bp DNA is correlated with less active genes compared with +1 H2A.Z nucleosomes with 120-140 bp DNA. We confirmed the unwrapping of H2A.Z nucleosomes under native condition by re-ChIP of H2A.Z and H2A after CTCF CUT&RUN in mouse ES cells. Importantly, we found that depletion of H2A.Z results in decreased unwrapping of H3.3 nucleosomes and increased CTCF binding. Taken together, through MNase-X-ChIP-seq, we showed that histone variant H2A.Z regulates nucleosome unwrapping in vivo and that its function in regulating transcription or CTCF binding is correlated with unwrapping states of H2A.Z nucleosomes.

Oncogene-induced telomere dysfunction enforces cellular senescence in human cancer precursor lesions.

In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans.

E2F transcription factor 1 regulates cellular and organismal senescence by inhibiting Forkhead box O transcription factors.

E2F1 and FOXO3 are two transcription factors that have been shown to participate in cellular senescence. Previous report reveals that E2F1 enhanced cellular senescence in human fibroblast cells, while FOXO transcription factors play against senescence by regulation reactive oxygen species scavenging proteins. However, their functional interplay has been unclear. Here we use E2F1 knock-out murine Embryonic fibroblasts (MEFs), knockdown RNAi constructs, and ectopic expression of E2F1 to show that it functions by negatively regulating FOXO3. E2F1 attenuates FOXO3-mediated expression of MnSOD and Catalase without affecting FOXO3 protein stability, subcellular localization, or phosphorylation by Akt. We mapped the interaction between E2F1 and FOXO3 to a region including the DNA binding domain of E2F1 and the C-terminal transcription-activation domain of FOXO3. We propose that E2F1 inhibits FOXO3-dependent transcription by directly binding FOXO3 in the nucleus and preventing activation of its target genes. Moreover, knockdown of the Caenorhabditis elegans E2F1 ortholog efl-1 significantly extends lifespan in a manner that requires the activity of the C. elegans FOXO gene daf-16. We conclude that there is an evolutionarily conserved signaling connection between E2F1 and FOXO3, which regulates cellular senescence and aging by regulating the activity of FOXO3. We speculate that drugs and/or therapies that inhibit this physical interaction might be good candidates for reducing cellular senescence and increasing longevity.

Analysis of Local Chromatin States Reveals Gene Transcription Potential during Mouse Neural Progenitor Cell Differentiation.

Chromatin dynamics play a critical role in cell fate determination and maintenance by regulating the expression of genes essential for development and differentiation. In mouse embryonic stem cells (mESCs), maintenance of pluripotency coincides with a poised chromatin state containing active and repressive histone modifications. However, the structural features of poised chromatin are largely uncharacterized. By adopting mild time-course MNase-seq with computational analysis, the low-compact chromatin in mESCs is featured in two groups: one in more open regions, corresponding to an active state, and the other enriched with bivalent histone modifications, considered the poised state. A parameter called the chromatin opening potential index (COPI) is also devised to quantify the transcription potential based on the dynamic changes of MNase-seq signals at promoter regions. Use of COPI provides effective prediction of gene activation potential and, more importantly, reveals a few developmental factors essential for mouse neural progenitor cell (NPC) differentiation.

The ATTCT repeats of spinocerebellar ataxia type 10 display strong nucleosome assembly which is enhanced by repeat interruptions.

Nucleosome packaging influences many aspects of DNA metabolism such as replication, repair and transcription, and via this link likely has further downstream effects on genome stability. The instability and expansion of repetitive sequences is associated with at least 42 human diseases, yet the molecular conditions contributing to repeat instability have remained largely undetermined. Previously we showed strong nucleosome formation on CAG repeats associated with spinocerebellar ataxia type 1 and very weak formation on CGG repeats associated with fragile X syndrome, and that interruption of these repeat tracts made the DNA behave more like random sequences. In this study, we determined nucleosome formation on pure and interrupted ATTCT pentanucleotides associated with spinocerebellar ataxia type 10 (SCA10). We report strong nucleosome formation on ATTCT repeats, like CAG tracts. Surprisingly, in contrast to the effect of interruptions on other repeat sequences, interruptions in the expanded ATTCT tracts further strengthened assembly with hyperacetylated histones under physiological conditions with NAP-1. These differences may contribute to phenotypic variation seen between families having pure and interrupted SCA10 repeats, as well as the overall genetic instability at the SCA10 locus.

Friedreich's ataxia GAA.TTC duplex and GAA.GAA.TTC triplex structures exclude nucleosome assembly.

Both chromatin structure and formation of triplex DNA at expanded GAA TTC repeats have been shown to regulate the FXN gene silencing, which causes Friedreich's ataxia. Recent studies have suggested that the presence of heterochromatin at the long expanded GAA TTC repeats, which is enriched in hypoacetylated histones, deters the transcription of the FXN gene. However, neither direct histone binding nor the effect of histone acetylation on the GAA TTC duplex or the GAA GAA TTC triplex has been measured in vitro. In this study, GAA TTC repeating DNAs derived from the human FXN gene, and the GAA GAA TTC triplex, were examined for their ability to assemble single nucleosomes and nucleosome arrays. Competitive nucleosome reconstitution assays demonstrated that the GAA TTC duplex excludes nucleosomes (53% decrease compared to the pUC control DNA) and that the GAA GAA TTC triplex further lowers the nucleosome assembly efficiency (82% decrease compared to the duplex DNA). The difference in assembly efficiency is amplified more significantly when hypoacetylated histones are used, compared to assembly with hyperacetylated histones. By analyzing the formation of nucleosome arrays on GAA TTC-containing plasmids, the triplex structure was shown to destabilize the ability of adjacent sequences to assemble nucleosomes. These results provide the first direct binding measurements for the GAA TTC duplex and the GAA GAA TTC triplex, and on the effect of histone acetylation, towards dissecting the role of chromatin structure in silencing of the FXN gene. These findings suggest that these sequences could profoundly alter local chromatin structure, and the discrepancy between in vivo and in vitro results supports recent studies showing that, in addition to DNA sequences, other factors such as epigenetic marks could be involved in the mechanism for inhibition of FXN gene expression.