Sirtuins are known to influence chromatin dynamics related to gene silencing, DNA repair, and maintenance of chromosome structural features such as telomeres. In this study we examined processes involving chromatin dynamics that are easily synchronized and monitored using Tetrahymena conjugation. Sirtuin inhibition with nicotinamide (NAM) affected three events requiring significant changes to global chromatin structure: (1) meiotic chromosome condensation, (2) differentiation of transcriptionally inert nuclei, and (3) programmed nuclear degradation.
During meiotic prophase, the germline micronucleus elongates ~50-fold resulting in a string-like "crescent" nucleus. Chromosomes decondense and assume a bouquet-like formation for homologous pairing and recombination, then re-condense prior to metaphase I [31
]. With 50 mM NAM treatment, decondensation and elongation was unaffected (normal numbers of fully elongated crescents were observed), but re-condensation appeared incomplete; the majority of cells arrested with partially elongated micronuclei (Figure ). The class I sirtuin, SIRT2, was previously implicated in promoting chromosome condensation during mitosis in human cells by deacetylating histone H4 [33
]. However, evidence for sirtuin roles in meiotic chromatin dynamics was lacking prior to this study.
Cells in a lower NAM concentration proceeded through meiosis, but displayed other chromatin differentiation defects. First, more than four post-zygotic nuclei were observed in most cells. Of these nuclei, all (or most in some cells) initiated new macronucleus chromatin development involving loss of micronuclear linker histone (MLH) (Figure ), and gain of both Nop52 (Figure ) and histone acetylation (Figure ). The ratio of differentiating macronuclei to micronuclei was 3-6:1 instead of the normal 2:1 in most pairs (Figures and -all parts). These data suggest that sirtuins normally promote the retention of MLH and development of heterochromatin in micronucleus-destined nuclei. Our observations suggest that the abnormally large number of nuclei in sirtuin-inhibited cells resulted from failure of gamete degradation prior to fertilization (3 of the 4 gametes normally degrade), an aberrant event that could be mechanistically related to the failure of parental macronuclear degradation observed later in conjugation. This idea is consistent with a previous observation that halting macronuclear degradation with PI-3 kinase inhibitors led to the retention and reprogramming of pronuclei to differentiate into micro- and macronuclei [34
]. Our results indicate that the retained extra nuclei are capable of differentiating from transcriptionally silent to transcriptionally active nuclei, and that this pathway is chosen (over maintenance of the silent state) in the absence of sirtuin activity.
Unlike normal cells that lose their parental macronucleus by 16 hours into conjugation, NAM-treated cells retained a macronucleus that exhibited little to no DNA degradation (Figure ). Programmed macronuclear degradation is thought to proceed through an apoptosis-like, caspase-independent mechanism that is dependent on endonuclease G activity and apoptosis inducing factor (AIF) stored in mitochondria [35
]. There is a well-described relationship in which chromatin condensation precedes DNA fragmentation in apoptosis [37
]; one that also applies to programmed nuclear death in Tetrahymena
], but the mechanistic details underlying these chromatin changes in any system are poorly understood. In Tetrahymena
, macronuclear DNA first condenses to less than half its original volume by 14 hours of conjugation, followed by global degradation of chromatin and resorption by the autophagosome [13
]. This degree of condensation was inhibited in the majority of NAM-treated cells (Figure ). Given that DNA degradation also failed in these nuclei (Figure ), sirtuin activity may be necessary for the chromatin condensation step of nuclear degradation, a prerequisite to global degradation. Interestingly, sirtuin inhibition did not affect the global deacetylation of histone H4 in parental macronuclear chromatin during pycnosis (Figure ), but it may be targeting other histones or nonhistone targets to mediate condensation. Our NAM treatment time course (Additional File 1
) suggests sirtuin involvement in an earlier degradation step, possibly involving initial signaling. Normally, commitment of nuclei to new macronuclear development at 6-7 hours into conjugation triggers destruction of the old parental macronucleus, which involves its migration to the posterior region of the cell [38
]. In our study, although NAM-treated nuclei committed to anlagen development, parental macronuclei failed to migrate in ~30% of cells (Figures and ) consistent with possible disruption of the degradation "triggering" event.
Sirtuins are involved in signaling pathways preventing apoptosis and cellular senescence in other organisms [41
], but no direct action on chromatin destined for degradation has been described. Although it is possible that the NAM treatment in our study inhibited nuclear degradation through blocking sirtuin-mediated signaling pathways, we present evidence that at least one Tetrahymena
sirtuin, Thd14, could act directly on chromatin destined for degradation. Thd14 selectively accumulated in the parental macronucleus (not in anlagen) at the initial stage of chromatin condensation (~8 hrs), and in later stages mapped precisely over the region of condensed chromatin within the macronuclear envelope (Figures and ). The mechanism for Thd14 accumulation will be the focus of future studies. We speculate that Thd14 plays a role in macronuclear degradation by directly modifying histones. While our HDAC assay confirmed that Thd14 is capable of deacetylating histones in vitro
(Additional File 3
), it is also possible that this enzyme may target other substrates under biologically relevant conditions. Other than histone H2AX and H2B phosphorylation, little is known about other modifications to apoptotic chromatin, but histone deacetylation appears critical in at least some cases. Deacetylation of yeast histone H2B by the Hos3 HDAC (class II enzyme) is required for apoptosis [42
], and apoptotic condensation in leukemia cells was linked with global histone deacetylation [43
], but the deacetylases remain unknown. Given their involvement in heterochromatin formation at various genomic loci [6
], sirtuins are reasonable candidates for apoptotic chromatin modifiers. Our combined results from NAM-inhibition and Thd14 localization provide the first evidence that a sirtuin(s) acts on chromatin destined for degradation.
One intriguing feature of Thd14 is its zinc finger domain, which is unusual to find on a sirtuin enzyme. Its strong homology to the PAZ domain on HDAC6 known to bind ubiquitin [23
], suggests that Thd14 may interact with ubiquitin or ubiquitinated proteins, especially since it contains all of the essential binding residues [24
] (Figure ). Since ubiquitin plays a major role in apoptosis and labels proteins for degradation, the PAZ and sirtuin domains of Thd14 may collaborate to make essential apoptotic modifications. Although a single protein with both a sirtuin and PAZ domain has not been identified in higher organisms, Thd14 may combine functions that higher-order organisms have evolved to handle with separate, more specialized proteins. To assess these possibilities, function of the putative PAZ domain and its potential role in nuclear degradation will be the subject of future studies.
Results in this study showed that Thd14 targeting was dependent on physiological state. In the nucleus of vegetatively growing cells, Thd14 resided primarily in the multiple nucleoli positioned around the nuclear periphery, and in mitochondria. Sirtuins in other organisms are known to act at nucleolar loci where they stabilize rDNA repeats and factor into RNA polymerase I transcription and rDNA silencing [44
]. Interestingly, nutrient starvation dramatically increased THD14
expression (Figure ), and caused Thd14 protein to concentrate into a prominent focus, or aggregate, inside the nucleus (Figure ). Like nucleoli, this aggregate was often associated with the nuclear periphery and stained weakly with DAPI (Figure , left panel). However, it was much larger than the typical nucleolar aggregates previously observed by electron microscopy [48
], and was ringed only around the periphery by the nucleolar protein Nop52 (Figure ). Previous work showed that under certain gene over-expression conditions (PML and p53) the human Sir2 homolog, SIRT1, is recruited to discrete nuclear foci with promyelocytic leukemia (PML) protein, in "PML bodies" where it deacetylates proteins such as p53 [50
]. Similarly, Thd14 may be sequestering with other nuclear proteins, possibly those involved in regulating cellular response to starvation stress.
Our study raises the intriguing possibility that the sirtuin Thd14 is specialized for the formation of irreversible heterochromatin functionally linked to parental macronucleus degradation, but not for reversible, facultative heterochromatin like meiotic chromatin in micronuclei. Despite the elevated expression in pre-meiotic stages, Thd14 did not localize to micronuclei at any point before or during meiosis, and formation of these two types of heterochromatin appears mechanistically different with respect to to histone modifications [51
]. Especially intriguing is that localization results suggest additional roles for Thd14 in nucleoli and mitochondria. Whether they relate to nuclear degradation mechanisms will be a focus of future studies. In one possible model consistent with the macronuclear autophagy process, Thd14 is delivered specifically to the parental macronucleus from mitochondria that fuse with the nucleus prior to degradation, a mechanism previously shown to deliver other degradation factors such as endonucleases and AIF [35
]. Such a model would explain the increased concentration of parental macronuclear Thd14 in the absence of increased expression (Figure ). Regardless of its concentration mechanism, we expect future work to define a role for Thd14 in promoting or coordinating macronuclear autophagy.