Telomere transcription in MLL-rearranged acute leukemia: increased levels of TERRA associate with lymphoid lineage and are independent of telomere length and ploidy

Telomere transcription into telomeric repeat-containing RNA (TERRA) is an integral component of all aspects of chromosome end protection consisting of telomerase- or recombination-dependent telomere elongation, telomere capping, and preservation of (sub)telomeric heterochromatin structure. The chromatin modifier and transcriptional regulator MLL associates with telomeres and regulates TERRA transcription in telomere length homeostasis and response to telomere dysfunction. MLL fusion proteins (MLL-FPs), the product of MLL rearrangements in leukemia, also associate with telomeric chromatin. However, an effect on telomere transcription in MLL-rearranged (MLL-r) leukemia has not yet been evaluated. Here, we show increased UUAGGG repeat-containing RNA levels in MLL-r acute lymphoblastic leukemia (ALL). MLL rearrangements do not affect telomere length and increased levels of UUAGGG repeat-containing RNA correlate with mean telomere length and reflect increased levels of TERRA. Also, increased levels of TERRA in MLL-r ALL occur in the presence of telomerase activity and are independent of ploidy, an underestimated source of variation on the overall transcriptome size in a cell. This MLL rearrangement-dependent and lymphoid lineage-associated increase in levels of TERRA supports a sustained telomere transcription by MLL-FPs that correlates with marked genomic stability previously reported in pediatric MLL-r ALL.

A role for Gcn5 in heterochromatin structure, gene silencing and NER at the HML locus in budding yeast

Histone acetyltransferase Gcn5 plays an important role in transcription activation, DNA replication-coupled nucleosome assembly and nucleotide excision repair (NER). However, its functions on the heterochromatin are unexplored. Here, we find that removal of Gcn5 leads to more condensed heterochromatin structure, as revealed by topology analysis of HML circles. Importantly, the altered heterochromatin structure is restored by re-expression of Gcn5 in the gcn5∆ cells. As a result of the more compact heterochromatin, gene silencing at the HML locus is increased and NER efficiency at HML is impaired in the absence of Gcn5. Interestingly, while the association of SIR complex with HML is enhanced in cells lacking Gcn5, the altered compaction of HML heterochromatin is also observed due to the deletion of Gcn5 from Sir-cells. These findings reveal a role of Gcn5 in the regulation of heterochromatin structure, gene silencing and NER efficiency at the heterochromatic HML locus in yeast.

The 20S proteasome activator PA28γ controls the compaction of chromatin

ABSTRACTPA28γ (also known as PSME3), a nuclear activator of the 20S proteasome, is involved in the degradation of several proteins regulating cell growth and proliferation and in the dynamics of various nuclear bodies, but its precise cellular functions remain unclear. Here, using a quantitative FLIM-FRET based microscopy assay monitoring close proximity between nucleosomes in living human cells, we show that PA28γ controls chromatin compaction. We find that its depletion induces a decompaction of pericentromeric heterochromatin, which is similar to what is observed upon the knockdown of HP1β (also known as CBX1), a key factor of the heterochromatin structure. We show that PA28γ is present at HP1β-containing repetitive DNA sequences abundant in heterochromatin and, importantly, that HP1β on its own is unable to drive chromatin compaction without the presence of PA28γ. At the molecular level, we show that this novel function of PA28γ is independent of its stable interaction with the 20S proteasome, and most likely depends on its ability to maintain appropriate levels of H3K9me3 and H4K20me3, histone modifications that are involved in heterochromatin formation. Overall, our results implicate PA28γ as a key factor involved in the regulation of the higher order structure of chromatin.

Metabolic regulation of telomere silencing by SESAME complex-catalyzed H3T11 phosphorylation

Telomeres are organized into a heterochromatin structure and maintenance of silent heterochromatin is required for chromosome stability. How telomere heterochromatin is dynamically regulated in response to stimuli remains unknown. Pyruvate kinase Pyk1 forms a complex named SESAME (Serine-responsive SAM-containing Metabolic Enzyme complex) to regulate gene expression by phosphorylating histone H3T11 (H3pT11). Here, we identify a function of SESAME in regulating telomere heterochromatin structure. SESAME phosphorylates H3T11 at telomeres, which maintains SIR (silent information regulator) complex occupancy at telomeres and protects Sir2 from degradation by autophagy. Moreover, SESAME-catalyzed H3pT11 directly represses autophagy-related gene expression to further prevent autophagy-mediated Sir2 degradation. By promoting H3pT11, serine increases Sir2 protein levels and enhances telomere silencing. Loss of H3pT11 leads to reduced Sir2 and compromised telomere silencing during chronological aging. Together, our study provides insights into dynamic regulation of silent heterochromatin by histone modifications and autophagy in response to cell metabolism and aging.

DNA polymerase epsilon is required for heterochromatin maintenance in Arabidopsis

Background Chromatin organizes DNA and regulates its transcriptional activity through epigenetic modifications. Heterochromatic regions of the genome are generally transcriptionally silent, while euchromatin is more prone to transcription. During DNA replication, both genetic information and chromatin modifications must be faithfully passed on to daughter strands. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear. Results We isolate a strong hypomorphic Arabidopsis thaliana mutant of the POL2A catalytic subunit of DNA polymerase epsilon and show that POL2A is required to stabilize heterochromatin silencing genome-wide, likely by preventing replicative stress. We reveal that POL2A inhibits DNA methylation and histone H3 lysine 9 methylation. Hence, the release of heterochromatin silencing in POL2A-deficient mutants paradoxically occurs in a chromatin context of increased levels of these two repressive epigenetic marks. At the nuclear level, the POL2A defect is associated with fragmentation of heterochromatin. Conclusion These results indicate that POL2A is critical to heterochromatin structure and function, and that unhindered replisome progression is required for the faithful propagation of DNA methylation throughout the cell cycle.

TELOMERE BIOLOGY: TELOMERE STRUCTURE, FUNCTION AND RELATED DISEASES

Telomere is the special heterochromatin structure which caps the end of eukaryotic chromosome and ensures the faithful replication of genetic materials. It also provides the protection against DNA damage signals and ensures the genome integrity and stability. Telomerase complete its task by its unique nucleoprotein structure. Here in this paper detail about nucleoprotein structure and their function is included. Robustness of function of telomere across cell cycle is guaranteed by the interaction between telomere and its interacting proteins. Recent findings regarding telomere biology and cancer are also included in this paper. KEY WORDS: Telomere, DNA damage, cell cycle, cancer

Swi6/HP1 binding to RNA-DNA hybrids initiates heterochromatin assembly

Heterochromatin formation in fission yeast and metazoans involves di/trimethylation of histone H3 at lysine 9 position (me2/me3-K9-H3) by the histone methyltransferase (HMT) Suv39/Clr4, followed by binding of Swi6/HP1 to me2/me3-K9-H3 via its chromodomain1. Subsequent self-association of Swi6/HP1 on adjacent nucleosomes leads to folded heterochromatin structure1-3. An alternate model suggests a concerted participation of Clr4 and Swi6/HP12,3. HP1 binding to RNA has been invoked for heterochromatin silencing in metazoans4,5. Swi6/HP1 also binds and channels RNA to exosome pathway in fission yeast6. Recruitment of Swi6/HP1 to centromere is also dependent on the RNAi pathway7. Here we show that Swi6/HP1 exhibits binding to RNAs, ranging from promiscuous, low-affinity binding to mRNAs, to moderate-affinity binding to the RNAi-generated siRNAs corresponding to the repeats present in heterochromatin regions7, to high affinity binding to the RNA-DNA hybrids cognate to the repeats. Together with sensitivity of Swi6 localization and silencing to RNaseH, our results suggest a dynamic distribution of Swi6/HP1 among the heterochromatin and euchromatic transcripts and binding to RNA-DNA hybrid as an RNAi-dependent and Me2/me3-K9-H3-independent mechanism of recruitment, leading to heterochromatin formation and silencing.

Identification of Genes Encoding CENP-A and Heterochromatin Protein 1 of Lipomyces starkeyi and Functional Analysis Using Schizosaccharomyces pombe

Centromeres function as a platform for the assembly of multiple kinetochore proteins and are essential for chromosome segregation. An active centromere is characterized by the presence of a centromere-specific histone H3 variant, CENP-A. Faithful centromeric localization of CENP-A is supported by heterochromatin in almost all eukaryotes; however, heterochromatin proteins have been lost in most Saccharomycotina. Here, identification of CENP-A (CENP-AL.s.) and heterochromatin protein 1 (Lsw1) in a Saccharomycotina species, the oleaginous yeast Lipomyces starkeyi, is reported. To determine if these proteins are functional, the proteins in S. pombe, a species widely used to study centromeres, were ectopically expressed. CENP-AL.s. localizes to centromeres and can be replaced with S. pombe CENP-A, indicating that CENP-AL.s. is a functional centromere-specific protein. Lsw1 binds at heterochromatin regions, and chromatin binding is dependent on methylation of histone H3 at lysine 9. In other species, self-interaction of heterochromatin protein 1 is thought to cause folding of chromatin, triggering transcription repression and heterochromatin formation. Consistent with this, it was found that Lsw1 can self-interact. L. starkeyi chromatin contains the methylation of histone H3 at lysine 9. These results indicated that L. starkeyi has a primitive heterochromatin structure and is an attractive model for analysis of centromere heterochromatin evolution.

DNA polymerase epsilon is a central coordinator of heterochromatin structure and function in Arabidopsis

BackgroundChromatin organizes the DNA molecule and regulates its transcriptional activity through epigenetic modifications. Heterochromatic regions of the genome are generally transcriptionally silent while euchromatin is more prone to transcription. During DNA replication, both genetic information and chromatin modifications must be faithfully passed on to daughter strands. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear.ResultsWe isolate a strong hypomorphic Arabidopsis thaliana mutant of the POL2A catalytic subunit of DNA polymerase epsilon and show that POL2A is required to stabilize heterochromatin silencing genome wide, likely by preventing replicative stress. We reveal that POL2A inhibits DNA methylation and histone H3 lysine 9 methylation. Hence, release of heterochromatin silencing in POL2A deficient mutants paradoxically occurs in a chromatin context of increased level of these two repressive epigenetic marks. At the nuclear level, POL2A defect is associated with fragmentation of heterochromatin.ConclusionThese results indicate that POL2A is critical to secure both heterochromatin structure and function. We also reveal that unhindered replisome progression is required for the faithful propagation of DNA methylation through the cell cycle.

Biogenesis of Developmental Master Regulatory 27nt-RNAs in Stylonychia—Can Coding RNA Turn into Non-Coding?

In the ciliate Stylonychia, somatic macronuclei differentiate from germline micronuclei during sexual reproduction, accompanied by developmental sequence reduction. Concomitantly, over 95% of micronuclear sequences adopt a heterochromatin structure characterized by the histone variant H3.4 and H3K27me3. RNAi-related genes and histone variants dominate the list of developmentally expressed genes. Simultaneously, 27nt-ncRNAs that match sequences retained in new macronuclei are synthesized and bound by PIWI1. Recently, we proposed a mechanistic model for ‘RNA-induced DNA replication interference’ (RIRI): during polytene chromosome formation PIWI1/27nt-RNA-complexes target macronucleus-destined sequences (MDS) by base-pairing and temporarily cause locally stalled replication. At polytene chromosomal segments with ongoing replication, H3.4K27me3-nucleosomes become selectively deposited, thus dictating the prospective heterochromatin structure of these areas. Consequently, these micronucleus-specific sequences become degraded, whereas 27nt-RNA-covered sites remain protected. However, the biogenesis of the 27nt-RNAs remains unclear. It was proposed earlier that in stichotrichous ciliates 27nt-RNA precursors could derive from telomere-primed bidirectional transcription of nanochromosomes and subsequent Dicer-like (DCL) activity. As a minimalistic explanation, we propose here that the 27nt-RNA precursor could rather be mRNA or pre-mRNA and that the transition of coding RNA from parental macronuclei to non-coding RNAs, which act in premature developing macronuclei, could involve RNA-dependent RNA polymerase (RDRP) activity creating dsRNA intermediates prior to a DCL-dependent pathway. Interestingly, by such mechanism the partition of a parental somatic genome and possibly also the specific nanochromosome copy numbers could be vertically transmitted to the differentiating nuclei of the offspring.