Lakxmi Subramanian

Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast

The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.

A histone H3K36 chromatin switch coordinates DNA double-strand break repair pathway choice

DNA double-strand break (DSB) repair is a highly regulated process performed predominantly by non-homologous end joining (NHEJ) or homologous recombination (HR) pathways. How these pathways are coordinated in the context of chromatin is unclear. Here we uncover a role for histone H3K36 modification in regulating DSB repair pathway choice in fission yeast. We find Set2-dependent H3K36 methylation reduces chromatin accessibility, reduces resection and promotes NHEJ, while antagonistic Gcn5-dependent H3K36 acetylation increases chromatin accessibility, increases resection and promotes HR. Accordingly, loss of Set2 increases H3K36Ac, chromatin accessibility and resection, while Gcn5 loss results in the opposite phenotypes following DSB induction. Further, H3K36 modification is cell cycle regulated with Set2-dependent H3K36 methylation peaking in G1 when NHEJ occurs, while Gcn5-dependent H3K36 acetylation peaks in S/G2 when HR prevails. These findings support an H3K36 chromatin switch in regulating DSB repair pathway choice.

Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres

To maintain genomic integrity, telomeres must undergo switches from a protected state to an accessible state that allows telomerase recruitment. To better understand how telomere accessibility is regulated in fission yeast, we analysed cell cycle‐dependent recruitment of telomere‐specific proteins (telomerase Trt1, Taz1, Rap1, Pot1 and Stn1), DNA replication proteins (DNA polymerases, MCM, RPA), checkpoint protein Rad26 and DNA repair protein Nbs1 to telomeres. Quantitative chromatin immunoprecipitation studies revealed that MCM, Nbs1 and Stn1 could be recruited to telomeres in the absence of telomere replication in S‐phase. In contrast, Trt1, Pot1, RPA and Rad26 failed to efficiently associate with telomeres unless telomeres are actively replicated. Unexpectedly, the leading strand DNA polymerase ε (Polε) arrived at telomeres earlier than the lagging strand DNA polymerases α (Polα) and δ (Polδ). Recruitment of RPA and Rad26 to telomeres matched arrival of DNA Polε, whereas S‐phase specific recruitment of Trt1, Pot1 and Stn1 matched arrival of DNA Polα. Thus, the conversion of telomere states involves an unanticipated intermediate step where lagging strand synthesis is delayed until telomerase is recruited.

Fission Yeast Tel1 ATM and Rad3 ATR Promote Telomere Protection and Telomerase Recruitment

The checkpoint kinases ATM and ATR are redundantly required for maintenance of stable telomeres in diverse organisms, including budding and fission yeasts, Arabidopsis, Drosophila, and mammals. However, the molecular basis for telomere instability in cells lacking ATM and ATR has not yet been elucidated fully in organisms that utilize both the telomere protection complex shelterin and telomerase to maintain telomeres, such as fission yeast and humans. Here, we demonstrate by quantitative chromatin immunoprecipitation (ChIP) assays that simultaneous loss of Tel1ATM and Rad3ATR kinases leads to a defect in recruitment of telomerase to telomeres, reduced binding of the shelterin complex subunits Ccq1 and Tpz1, and increased binding of RPA and homologous recombination repair factors to telomeres. Moreover, we show that interaction between Tpz1-Ccq1 and telomerase, thought to be important for telomerase recruitment to telomeres, is disrupted in tel1Δ rad3Δ cells. Thus, Tel1ATM and Rad3ATR are redundantly required for both protection of telomeres against recombination and promotion of telomerase recruitment. Based on our current findings, we propose the existence of a regulatory loop between Tel1ATM/Rad3ATR kinases and Tpz1-Ccq1 to ensure proper protection and maintenance of telomeres in fission yeast.

Recombination-Based Telomere Maintenance Is Dependent on Tel1-MRN and Rap1 and Inhibited by Telomerase, Taz1, and Ku in Fission Yeast

ABSTRACT Fission yeast cells survive loss of the telomerase catalytic subunit Trt1 (TERT) through recombination-based telomere maintenance or through chromosome circularization. Although trt1Δ survivors with linear chromosomes can be obtained, they often spontaneously circularize their chromosomes. Therefore, it was difficult to establish genetic requirements for telomerase-independent telomere maintenance. In contrast, when the telomere-binding protein Taz1 is also deleted, taz1Δ trt1Δ cells are able to stably maintain telomeres. Thus, taz1Δ trt1Δ cells can serve as a valuable tool in understanding the regulation of telomerase-independent telomere maintenance. In this study, we show that the checkpoint kinase Tel1 (ATM) and the DNA repair complex Rad32-Rad50-Nbs1 (MRN) are required for telomere maintenance in taz1Δ trt1Δ cells. Surprisingly, Rap1 is also essential for telomere maintenance in taz1Δ trt1Δ cells, even though recruitment of Rap1 to telomeres depends on Taz1. Expression of catalytically inactive Trt1 can efficiently inhibit recombination-based telomere maintenance, but the inhibition requires both Est1 and Ku70. While Est1 is essential for recruitment of Trt1 to telomeres, Ku70 is dispensable. Thus, we conclude that Taz1, TERT-Est1, and Ku70-Ku80 prevent telomere recombination, whereas MRN-Tel1 and Rap1 promote recombination-based telomere maintenance. Evolutionarily conserved proteins in higher eukaryotic cells might similarly contribute to telomere recombination.

Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly.

CENP-A chromatin forms the foundation for kinetochore assembly. Replication-independent incorporation of CENP-A at centromeres depends on its chaperone HJURPScm3, and Mis18 in vertebrates and fission yeast. The recruitment of Mis18 and HJURPScm3 to centromeres is cell cycle regulated. Vertebrate Mis18 associates with Mis18BP1KNL2, which is critical for the recruitment of Mis18 and HJURPScm3. We identify two novel fission yeast Mis18-interacting proteins (Eic1 and Eic2), components of the Mis18 complex. Eic1 is essential to maintain Cnp1CENP-A at centromeres and is crucial for kinetochore integrity; Eic2 is dispensable. Eic1 also associates with Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21, components of the constitutive CCAN/Mis6/Ctf19 complex. No Mis18BP1KNL2 orthologue has been identified in fission yeast, consequently it remains unknown how the key Cnp1CENP-A loading factor Mis18 is recruited. Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1KNL2, thus representing the functional counterpart of Mis18BP1KNL2 in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3HJURP Cnp1CENP-A loading factors. The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.

Roles of Heterochromatin and Telomere Proteins in Regulation of Fission Yeast Telomere Recombination and Telomerase Recruitment

When the telomerase catalytic subunit (Trt1/TERT) is deleted, a majority of fission yeast cells survives by circularizing chromosomes. Alternatively, a small minority survives by maintaining telomeric repeats through recombination among telomeres. The recombination-based telomere maintenance in trt1Δ cells is inhibited by the telomere protein Taz1. In addition, catalytically inactive full-length Trt1 (Trt1-CI) and truncated Trt1 lacking the T-motif and reverse transcriptase (RT) domain (Trt1-ΔT/RT) can strongly inhibit recombination-based survival. Here, we investigated the effects of deleting the heterochromatin proteins Swi6 (HP1 ortholog) and Clr4 (Suv39 family of histone methyltransferases) and the telomere capping complex subunits Poz1 and Ccq1 on Taz1- and Trt1-dependent telomere recombination inhibition. The ability of Taz1 to inhibit telomere recombination did not require Swi6, Clr4, Poz1, or Ccq1. Although Swi6, Clr4, and Poz1 were dispensable for the inhibition of telomere recombination by Trt1-CI, Ccq1 was required for efficient telomere recruitment of Trt1 and Trt1-CI-dependent inhibition of telomere recombination. We also found that Swi6, Clr4, Ccq1, the checkpoint kinase Rad3 (ATR ortholog), and the telomerase regulatory subunit Est1 are all required for Trt1-ΔT/RT to inhibit telomere recombination. However, because loss of Swi6, Clr4, Rad3, Ccq1, or Est1 did not significantly alter the recruitment efficiency of Trt1-ΔT/RT to telomeres, these factors are likely to enhance the ability of Trt1-ΔT/RT to inhibit recombination-based survival by contributing to the negative regulation of telomere recombination.

Centromere localization and function of Mis18 requires Yippee‐like domain‐mediated oligomerization

Mis18 is a key regulator responsible for the centromere localization of the CENP‐A chaperone Scm3 in Schizosaccharomyces pombe and HJURP in humans, which establishes CENP‐A chromatin that defines centromeres. The molecular and structural determinants of Mis18 centromere targeting remain elusive. Here, by combining structural, biochemical, and yeast genetic studies, we show that the oligomerization of S. pombe Mis18, mediated via its conserved N‐terminal Yippee‐like domain, is crucial for its centromere localization and function. The crystal structure of the N‐terminal Yippee‐like domain reveals a fold containing a cradle‐shaped pocket that is implicated in protein/nucleic acid binding, which we show is required for Mis18 function. While the N‐terminal Yippee‐like domain forms a homodimer in vitro and in vivo, full‐length Mis18, including the C‐terminal α‐helical domain, forms a homotetramer in vitro. We also show that the Yippee‐like domains of human Mis18α/Mis18β interact to form a heterodimer, implying a conserved structural theme for Mis18 regulation.

A Kinase-Independent Role for the Rad3ATR-Rad26ATRIP Complex in Recruitment of Tel1ATM to Telomeres in Fission Yeast

ATM and ATR are two redundant checkpoint kinases essential for the stable maintenance of telomeres in eukaryotes. Previous studies have established that MRN (Mre11-Rad50-Nbs1) and ATRIP (ATR Interacting Protein) interact with ATM and ATR, respectively, and recruit their partner kinases to sites of DNA damage. Here, we investigated how Tel1ATM and Rad3ATR recruitment to telomeres is regulated in fission yeast. Quantitative chromatin immunoprecipitation (ChIP) assays unexpectedly revealed that the MRN complex could also contribute to the recruitment of Tel1ATM to telomeres independently of the previously established Nbs1 C-terminal Tel1ATM interaction domain. Recruitment of Tel1ATM to telomeres in nbs1-c60Δ cells, which lack the C-terminal 60 amino acid Tel1ATM interaction domain of Nbs1, was dependent on Rad3ATR-Rad26ATRIP, but the kinase domain of Rad3ATR was dispensable. Thus, our results establish that the Rad3ATR-Rad26ATRIP complex contributes to the recruitment of Tel1ATM independently of Rad3ATR kinase activity, by a mechanism redundant with the Tel1ATM interaction domain of Nbs1. Furthermore, we found that the N-terminus of Nbs1 contributes to the recruitment of Rad3ATR-Rad26ATRIP to telomeres. In response to replication stress, mammalian ATR–ATRIP also contributes to ATM activation by a mechanism that is dependent on the MRN complex but independent of the C-terminal ATM interaction domain of Nbs1. Since telomere protection and DNA damage response mechanisms are very well conserved between fission yeast and mammalian cells, mammalian ATR–ATRIP may also contribute to the recruitment of ATM to telomeres and to sites of DNA damage independently of ATR kinase activity.

Roles of the Checkpoint Sensor Clamp Rad9-Rad1-Hus1 (911)-Complex and the Clamp Loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe Telomere Maintenance

While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.