Silent Mating-type Loci Research Articles

The histone chaperone FACT facilitates heterochromatin spreading by regulating histone turnover and H3K9 methylation states.

SUMMARYHeterochromatin formation requires three distinct steps: nucleation, self-propagation (spreading) along the chromosome, and faithful maintenance after each replication cycle. Impeding any of those steps induces heterochromatin defects and improper gene expression. The essential histone chaperone FACT (facilitates chromatin transcription) has been implicated in heterochromatin silencing, but the mechanisms by which FACT engages in this process remain opaque. Here, we pinpoint its function to the heterochromatin spreading process in fission yeast. FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes in the vicinity of heterochromatin boundaries. FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading. FACT mutant spreading defects are suppressed by removal of the H3K9 methylation antagonist Epe1. Together, our study identifies FACT as a histone chaperone that promotes heterochromatin spreading and lends support to the model that regulated histone turnover controls the propagation of repressive methylation marks.

A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata

Mating-type switching is a complex mechanism that promotes sexual reproduction in Saccharomycotina. In the model species Saccharomyces cerevisiae, mating-type switching is initiated by the Ho endonuclease that performs a site-specific double-strand break (DSB) at MAT, repaired by homologous recombination (HR) using one of the two silent mating-type loci, HMLalpha and HMRa. The reasons why all the elements of the mating-type switching system have been conserved in some Saccharomycotina, that do not show a sexual cycle nor mating-type switching, remain unknown. To gain insight on this phenomenon, we used the yeast Candida glabrata, phylogenetically close to S. cerevisiae, and for which no spontaneous and efficient mating-type switching has been observed. We have previously shown that expression of S. cerevisiae's Ho (ScHo) gene triggers mating-type switching in C. glabrata, but this leads to massive cell death. In addition, we unexpectedly found, that not only MAT but also HML was cut in this species, suggesting the formation of multiple chromosomal DSBs upon HO induction. We now report that HMR is also cut by ScHo in wild-type strains of C. glabrata. To understand the link between mating-type switching and cell death in C. glabrata, we constructed strains mutated precisely at the Ho recognition sites. We find that even when HML and HMR are protected from the Ho-cut, introducing a DSB at MAT is sufficient to induce cell death, whereas one DSB at HML or HMR is not. We demonstrate that mating-type switching in C. glabrata can be triggered using CRISPR-Cas9, without high lethality. We also show that switching is Rad51-dependent, as in S. cerevisiae, but that donor preference is not conserved in C. glabrata. Altogether, these results suggest that a DSB at MAT can be repaired by HR in C. glabrata, but that repair is prevented by ScHo.

Yeast mismatch repair components are required for stable inheritance of gene silencing.

Alterations in epigenetic silencing have been associated with ageing and tumour formation. Although substantial efforts have been made towards understanding the mechanisms of gene silencing, novel regulators in this process remain to be identified. To systematically search for components governing epigenetic silencing, we developed a genome-wide silencing screen for yeast (Saccharomyces cerevisiae) silent mating type locus HMR. Unexpectedly, the screen identified the mismatch repair (MMR) components Pms1, Mlh1, and Msh2 as being required for silencing at this locus. We further found that the identified genes were also required for proper silencing in telomeres. More intriguingly, the MMR mutants caused a redistribution of Sir2 deacetylase, from silent mating type loci and telomeres to rDNA regions. As a consequence, acetylation levels at histone positions H3K14, H3K56, and H4K16 were increased at silent mating type loci and telomeres but were decreased in rDNA regions. Moreover, knockdown of MMR components in human HEK293T cells increased subtelomeric DUX4 gene expression. Our work reveals that MMR components are required for stable inheritance of gene silencing patterns and establishes a link between the MMR machinery and the control of epigenetic silencing.

The Sir4 H-BRCT domain interacts with phospho-proteins to sequester and repress yeast heterochromatin.

In Saccharomyces cerevisiae, the silent information regulator (SIR) proteins Sir2/3/4 form a complex that suppresses transcription in subtelomeric regions and at the homothallic mating-type (HM) loci. Here, we identify a non-canonical BRCA1 C-terminal domain (H-BRCT) in Sir4, which is responsible for tethering telomeres to the nuclear periphery. We show that Sir4 H-BRCT and the closely related Dbf4 H-BRCT serve as selective phospho-epitope recognition domains that bind to a variety of phosphorylated target peptides. We present detailed structural information about the binding mode of established Sir4 interactors (Esc1, Ty5, Ubp10) and identify several novel interactors of Sir4 H-BRCT, including the E3 ubiquitin ligase Tom1. Based on these findings, we propose a phospho-peptide consensus motif for interaction with Sir4 H-BRCT and Dbf4 H-BRCT. Ablation of the Sir4 H-BRCT phospho-peptide interaction disrupts SIR-mediated repression and perinuclear localization. In conclusion, the Sir4 H-BRCT domain serves as a hub for recruitment of phosphorylated target proteins to heterochromatin to properly regulate silencing and nuclear order.

Modulation of Gene Silencing by Cdc7p via H4 K16 Acetylation and Phosphorylation of Chromatin Assembly Factor CAF-1 in Saccharomyces cerevisiae.

CAF-1 is an evolutionarily conserved H3/H4 histone chaperone that plays a key role in replication-coupled chromatin assembly and is targeted to the replication fork via interactions with PCNA, which, if disrupted, leads to epigenetic defects. In Saccharomyces cerevisiae, when the silent mating-type locus HMR contains point mutations within the E silencer, Sir protein association and silencing is lost. However, mutation of CDC7, encoding an S-phase-specific kinase, or subunits of the H4 K16-specific acetyltransferase complex SAS-I, restore silencing to this crippled HMR, HMRae** Here, we observed that loss of Cac1p, the largest subunit of CAF-1, also restores silencing at HMRae**, and silencing in both cac1Δ and cdc7 mutants is suppressed by overexpression of SAS2 We demonstrate Cdc7p and Cac1p interact in vivo in S phase, but not in G1, consistent with observed cell cycle-dependent phosphorylation of Cac1p, and hypoacetylation of chromatin at H4 K16 in both cdc7 and cac1Δ mutants. Moreover, silencing at HMRae** is restored in cells expressing cac1p mutants lacking Cdc7p phosphorylation sites. We also discovered that cac1Δ and cdc7-90 synthetically interact negatively in the presence of DNA damage, but that Cdc7p phosphorylation sites on Cac1p are not required for responses to DNA damage. Combined, our results support a model in which Cdc7p regulates replication-coupled histone modification via a CAC1-dependent mechanism involving H4 K16ac deposition, and thereby silencing, while CAF-1-dependent replication- and repair-coupled chromatin assembly per se are functional in the absence of phosphorylation of Cdc7p consensus sites on CAF-1.

Elevated dosage of Ulp1 disrupts telomeric silencing in Saccharomyces cerevisiae.

Heterochromatin in Saccharomyces cerevisiae is found at the telomeres and silent mating type loci. Many sub-telomeric loci are naturally silenced by this mechanism. In addition, when euchromatic genes are placed proximal to telomeric repeats they are subjected to heritable gene silencing that is referred to as telomere position effect. Establishment and maintenance of TPE is dependent on the assembly of silent information regulator proteins at these loci. Here we show that dosage of SUMO isopeptidase, Ulp1, is important for regulation of TPE. Moderate elevation of Ulp1 reduces silencing of both, the euchromatic gene placed proximal to telomeric repeats and the sub-telomeric genes that are silenced by TPE. We further demonstrate that this loss in silencing is due to reduced recruitment of one of the silent information regulators, Sir3p. We show that SUMO peptidase, Ulp1, regulates telomeric position effect by regulating the recruitment of Sir proteins.

DNA damaging agents trigger the expression of the HML silent mating type locus in Saccharomyces cerevisiae

Many DNA damaging agents also react with RNA and protein, and could thus cause epigenetic as well as genotoxic changes. To investigate which DNA damaging agents alter epigenetic states, we studied the chemical-induced changes in expression of the yeast silent mating type locus HMLα, which can be triggered by inhibiting yeast Sir2. We observed that the alkylating agent methyl methane sulfonate (MMS) can result in HMLα expression, using a colony sector assay that results from expression of a HML-positioned cre gene. Using single-cell imaging we also observed that alkylating agents, including MMS and methyl-3-nitro-1-nitrosoguanidine (MNNG), as well as short-wave UV, also decreased HML silencing. We suggest that chemical-induced alterations in heterochromatin structure could confer transient phenotypic changes that affect the cellular responses to DNA damaging agents.

Identification of a transcriptional activation domain in yeast repressor activator protein 1 (Rap1) using an altered DNA-binding specificity variant

Repressor activator protein 1 (Rap1) performs multiple vital cellular functions in the budding yeast Saccharomyces cerevisiae These include regulation of telomere length, transcriptional repression of both telomere-proximal genes and the silent mating type loci, and transcriptional activation of hundreds of mRNA-encoding genes, including the highly transcribed ribosomal protein- and glycolytic enzyme-encoding genes. Studies of the contributions of Rap1 to telomere length regulation and transcriptional repression have yielded significant mechanistic insights. However, the mechanism of Rap1 transcriptional activation remains poorly understood because Rap1 is encoded by a single copy essential gene and is involved in many disparate and essential cellular functions, preventing easy interpretation of attempts to directly dissect Rap1 structure-function relationships. Moreover, conflicting reports on the ability of Rap1-heterologous DNA-binding domain fusion proteins to serve as chimeric transcriptional activators challenge use of this approach to study Rap1. Described here is the development of an altered DNA-binding specificity variant of Rap1 (Rap1AS). We used Rap1AS to map and characterize a 41-amino acid activation domain (AD) within the Rap1 C terminus. We found that this AD is required for transcription of both chimeric reporter genes and authentic chromosomal Rap1 enhancer-containing target genes. Finally, as predicted for a bona fide AD, mutation of this newly identified AD reduced the efficiency of Rap1 binding to a known transcriptional coactivator TFIID-binding target, Taf5. In summary, we show here that Rap1 contains an AD required for Rap1-dependent gene transcription. The Rap1AS variant will likely also be useful for studies of the functions of Rap1 in other biological pathways.

Variants of the Sir4 Coiled-Coil Domain Improve Binding to Sir3 for Heterochromatin Formation in Saccharomyces cerevisiae.

Heterochromatin formation in the yeast Saccharomyces cerevisiae is characterized by the assembly of the Silent Information Regulator (SIR) complex, which consists of the histone deacetylase Sir2 and the structural components Sir3 and Sir4, and binds to unmodified nucleosomes to provide gene silencing. Sir3 contains an AAA+ ATPase-like domain, and mutations in an exposed loop on the surface of this domain abrogate Sir3 silencing function in vivo, as well in vitro binding to the Sir2/Sir4 subcomplex. Here, we found that the removal of a single methyl group in the C-terminal coiled-coil domain (mutation T1314S) of Sir4 was sufficient to restore silencing at the silent mating-type loci HMR and HML to a Sir3 version with a mutation in this loop. Restoration of telomeric silencing required further mutations of Sir4 (E1310V and K1325R). Significantly, these mutations in Sir4 restored in vitro complex formation between Sir3 and the Sir4 coiled-coil, indicating that the improved affinity between Sir3 and Sir4 is responsible for the restoration of silencing. Altogether, these observations highlight remarkable properties of selected amino-acid changes at the Sir3-Sir4 interface that modulate the affinity of the two proteins.

Set3 contributes to heterochromatin integrity by promoting transcription of subunits of Clr4-Rik1-Cul4 histone methyltransferase complex in fission yeast.

Heterochromatin formation in fission yeast depends on RNAi machinery and histone-modifying enzymes. One of the key histone-modifying complexes is Clr4-Rik1-Cul4 methyltransferase complex (CLRC), which mediates histone H3K9 methylation, a hallmark for heterochromatin. CLRC is composed of the Clr4 histone methyltransferase, Rik1, Raf1, Raf2 and Pcu4. However, transcriptional regulation of the CLRC subunits is not well understood. In this study, we identified Set3, a core subunit of the Set3/Hos2 histone deacetylase complex (Set3C), as a contributor to the integrity and silencing of heterochromatin at centromeres, telomeres and silent mating-type locus. This novel role of Set3 relies on its PHD finger, but is independent of deacetylase activity or structural integrity of Set3C. Set3 is not located to the centromeric region. Instead, Set3 is targeted to the promoters of clr4+ and rik1+, probably through its PHD finger. Set3 promotes transcription of clr4+ and rik1+. Consistently, the protein levels of Clr4 and Rik1 were reduced in the set3Δ mutant. The heterochromatin silencing defect in the set3Δ mutant could be rescued by overexpressing of clr4+ or rik1+. Our study suggests transcriptional activation of essential heterochromatin factors underlies the tight regulation of heterochromatin integrity.

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae.

Transcriptional silencing in Saccharomyces cerevisiae occurs at several genomic sites including the silent mating-type loci, telomeres, and the ribosomal DNA (rDNA) tandem array. Epigenetic silencing at each of these domains is characterized by the absence of nearly all histone modifications, including most prominently the lack of histone H4 lysine 16 acetylation. In all cases, silencing requires Sir2, a highly-conserved NAD(+)-dependent histone deacetylase. At locations other than the rDNA, silencing also requires additional Sir proteins, Sir1, Sir3, and Sir4 that together form a repressive heterochromatin-like structure termed silent chromatin. The mechanisms of silent chromatin establishment, maintenance, and inheritance have been investigated extensively over the last 25 years, and these studies have revealed numerous paradigms for transcriptional repression, chromatin organization, and epigenetic gene regulation. Studies of Sir2-dependent silencing at the rDNA have also contributed to understanding the mechanisms for maintaining the stability of repetitive DNA and regulating replicative cell aging. The goal of this comprehensive review is to distill a wide array of biochemical, molecular genetic, cell biological, and genomics studies down to the "nuts and bolts" of silent chromatin and the processes that yield transcriptional silencing.

‐Altered DNA‐Binding Specificity Transcription Activator Variant Provides Key To Unlocking Gene Activation Mechanism

Transcription activator proteins confer target selective, gene‐specific activation in response to appropriate stimuli. In order to dissect the molecular mechanisms of gene transcription activation, it is imperative to understand the role(s) of the transcription activator. Unfortunately, a subset of DNA‐binding transcriptional activator proteins are resistant to study by the conventional means of fusing putative Activation Domains (ADs) to heterologous DNA Binding Domains (DBDs) to identify key transcription factor functional elements. The budding yeast Saccharomyces cerevisiae Repressor Activator Protein 1 (Rap1) is one such activator protein. Rap1 specifically binds over 500 DNA sites in the yeast genome, serving key roles in multiple cellular processes including: (a) telomere stabilization; (b) transcription repression of silent mating type loci; and (c) activation of two of the most vigorously transcribed classes of yeast genes: the SAGA‐dominated glycolytic enzyme‐encoding genes (GEs) and the TFIID‐dominated ribosomal protein genes (RPGs). However, fusions of Rap1 or Rap1‐domains with heterologous DBDs reportedly fail to robustly drive reporter gene expression.To enable study of the role of Rap1 in activation, an altered DNA‐binding specificity Rap1 variant (Rap1 AS) was generated using a direct screening/selection strategy in yeast wherein activation of a selectable HIS3 reporter gene depended upon the ability of DBD‐mutagenized variant forms of Rap1 to efficiently drive expression from a mutant Rap1 enhancer (UASRAP1). Multiple candidate putative Rap1 AS variants scored in this screen. One has been characterized in detail. In both biochemical (DNA gel mobility shift) and functional (HIS3 reporter gene expression) assays, Rap1 AS preferentially bound the selecting mutant UASRAP1, displaying a significant discrimination for mutant vs. wild‐type (WT) UASRAP1 sequences. Yeast pseudodiploid for Rap1 (Rap1 WT and Rap1 AS) grow like WT, showing that Rap1 AS does not interfere with the many essential functions of Rap1 WT. Subsequent mutagenesis experiments with Rap1 AS identified a putative activation domain that mapped to a ~40 amino acid region within the C‐terminal 1/3 of Rap1. Systematic point mutagenesis of this mapped AD has identified eight amino acids that contribute critically to AD function.The collection of Rap1 AD mutants will be used to investigate the transcription activation mechanisms used to drive the TFIID‐dominated RPGs. Since we have previously shown that Rap1 interacts directly with TFIID, and is the only RPG activator absolutely required for RPG transcription, mechanistic analyses performed with the Rap1 AD mutants should unlock the key step(s) to RPG activation. Because few TFIID‐dependent activation mechanisms have been carefully dissected, these key steps will likely reveal general principles of TFIID‐dependent gene activation applicable to all organisms.Support or Funding InformationThis research was supported by the NIH Molecular Endocrinology Training Program (METP) training grant (T32 DK07563) and the NIH R01 grants GM52461 and GM115892.

A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5’-3’ Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe

Epigenetic gene silencing plays a critical role in regulating gene expression and contributes to organismal development and cell fate acquisition in eukaryotes. In fission yeast, Schizosaccharomyces pombe, heterochromatin-associated gene silencing is known to be mediated by RNA processing pathways including RNA interference (RNAi) and a 3’-5’ exoribonuclease complex, the exosome. Here, we report a new RNA-processing pathway that contributes to epigenetic gene silencing and assembly of heterochromatin mediated by 5’-3’ exoribonuclease Dhp1/Rat1/Xrn2. Dhp1 mutation causes defective gene silencing both at peri-centromeric regions and at the silent mating type locus. Intriguingly, mutation in either of the two well-characterized Dhp1-interacting proteins, the Din1 pyrophosphohydrolase or the Rhn1 transcription termination factor, does not result in silencing defects at the main heterochromatic regions. We demonstrate that Dhp1 interacts with heterochromatic factors and is essential in the sequential steps of establishing silencing in a manner independent of both RNAi and the exosome. Genomic and genetic analyses suggest that Dhp1 is involved in post-transcriptional silencing of repetitive regions through its RNA processing activity. The results describe the unexpected role of Dhp1/Rat1/Xrn2 in chromatin-based silencing and elucidate how various RNA-processing pathways, acting together or independently, contribute to epigenetic regulation of the eukaryotic genome.

The Conformation of Yeast Chromosome III Is Mating Type Dependent and Controlled by the Recombination Enhancer.

Mating-type switching in yeast occurs through gene conversion between the MAT locus and one of two silent loci (HML or HMR) on opposite ends of the chromosome. MATa cells choose HML as template, whereas MATα cells use HMR. The recombination enhancer (RE) located on the left arm regulates this process. One long-standing hypothesis is that switching is guided by mating-type-specific and possibly RE-dependent chromosome folding. Here, we use Hi-C, 5C, and live-cell imaging to characterize the conformation of chromosome III in both mating types. We discovered a mating-type-specific conformational difference in the left arm. Deletion of a 1-kb subregion within the RE, which is not necessary during switching, abolished mating-type-dependent chromosome folding. The RE is therefore a composite element with one subregion essential for donor selection during switching and a separate region involved in modulating chromosome conformation.

Heat stress-induced Cup9-dependent transcriptional regulation of SIR2.

The epigenetic writer Sir2 maintains the heterochromatin state of chromosome in three chromosomal regions, namely, the silent mating type loci, telomeres, and the ribosomal DNA (rDNA). In this study, we demonstrated the mechanism by which Sir2 is regulated under heat stress. Our study reveals that a transient heat shock causes a drastic reduction in the SIR2 transcript which results in sustained failure to initiate silencing for as long as 90 generations. Hsp82 overexpression, which is the usual outcome of heat shock treatment, leads to a similar downregulation of SIR2 transcription. Using a series of genetic experiments, we have established that heat shock or Hsp82 overexpression causes upregulation of CUP9 that, in turn, represses SIR2 transcription by binding to its upstream activator sequence. We have mapped the cis regulatory element of SIR2. Our study shows that the deletion of cup9 causes reversal of the Hsp82 overexpression phenotype and upregulation of SIR2 expression in heat-induced Hsp82-overexpressing cells. On the other hand, we found that Cup9 overexpression represses SIR2 transcription and leads to a failure in the establishment of heterochromatin. The results of our study highlight the mechanism by which environmental factors amend the epigenetic configuration of chromatin.

Multifaceted genome control by Set1 Dependent and Independent of H3K4 methylation and the Set1C/COMPASS complex.

Histone modifiers are critical regulators of chromatin-based processes in eukaryotes. The histone methyltransferase Set1, a component of the Set1C/COMPASS complex, catalyzes the methylation at lysine 4 of histone H3 (H3K4me), a hallmark of euchromatin. Here, we show that the fission yeast Schizosaccharomyces pombe Set1 utilizes distinct domain modules to regulate disparate classes of repetitive elements associated with euchromatin and heterochromatin via H3K4me-dependent and -independent pathways. Set1 employs its RNA-binding RRM2 and catalytic SET domains to repress Tf2 retrotransposons and pericentromeric repeats while relying on its H3K4me function to maintain transcriptional repression at the silent mating type (mat) locus and subtelomeric regions. These repressive functions of Set1 correlate with the requirement of Set1C components to maintain repression at the mat locus and subtelomeres while dispensing Set1C in repressing Tf2s and pericentromeric repeats. We show that the contributions of several Set1C subunits to the states of H3K4me diverge considerably from those of Saccharomyces cerevisiae orthologs. Moreover, unlike S. cerevisiae, the regulation of Set1 protein level is not coupled to the status of H3K4me or histone H2B ubiquitination by the HULC complex. Intriguingly, we uncover a genome organization role for Set1C and H3K4me in mediating the clustering of Tf2s into Tf bodies by antagonizing the acetyltransferase Mst1-mediated H3K4 acetylation. Our study provides unexpected insights into the regulatory intricacies of a highly conserved chromatin-modifying complex with diverse roles in genome control.

Solution-state conformation and stoichiometry of yeast Sir3 heterochromatin fibres.

Heterochromatin is a repressive chromatin compartment essential for maintaining genomic integrity. A hallmark of heterochromatin is the presence of specialized nonhistone proteins that alter chromatin structure to inhibit transcription and recombination. It is generally assumed that heterochromatin is highly condensed. However, surprisingly little is known about the structure of heterochromatin or its dynamics in solution. In budding yeast, formation of heterochromatin at telomeres and the HM silent mating type loci require the Sir3 protein. Here, we use a combination of sedimentation velocity, atomic force microscopy, and nucleosomal array capture to characterize the stoichiometry and conformation of Sir3 nucleosomal arrays. The results indicate that Sir3 interacts with nucleosomal arrays with a stoichiometry of two Sir3 monomers per nucleosome. We also find that Sir3 fibers are less compact than canonical – magnesium-induced 30 nm fibers. We suggest that heterochromatin proteins promote silencing by “coating” nucleosomal arrays, stabilizing interactions between nucleosomal histones and DNA.

CHANGES IN THE GENOME-WIDE LOCALIZATION PATTERN OF SIR3 IN SACCHAROMYCES CEREVISIAE DURING DIFFERENT GROWTH STAGES

In budding yeast, the Sir2, Sir3 and Sir4 proteins form SIR complexes, required for the assembly of silent heterochromatin domains, and which mediate transcription silencing at the telomeres as well as at silent mating type loci. In this study, under fluorescence microscopy, we found most Sir3-GFP expressions in the logarithmic phase cells appeared as multiple punctations as expected. However, some differences in the distribution of fluorescent signals were detected in the diauxic∼early stationary phase cells. To clarify these, we then used ChIP on chip assays to investigate the genome-wide localization of Sir3. In general, Sir3 binds to all 32 telomere proximal regions, the silent mating type loci and also binds to the rDNA region. However, the genome-wide localization patterns of Sir3 are different between these two distinct growth phases. We also confirmed that Sir3 binds to a recently identified secondary binding site, PAU genes, and further identified 349 Sir3-associated cluster regions. These results provide additional support in roles for Sir3 in the modulation of gene expression during physical conditions such as diauxic∼early stationary phase growing. Moreover, they imply that Sir3 may be not only involved in the formation of conventional silent heterochromatin, but also able to associate with some other chromatin regions involved in epigenetic regulation.

Comparative study of the promotion of porcine fetal fibroblast proliferation by overexpression of two transcriptional variants of SIRT6

Mammalian Sirtuin 6 (SIRT6) plays an important role in the transcriptional silencing of silent mating-type loci, telomeres, and rDNA. However, the roles of porcine SIRT6 in cell proliferation are poorly understood, and a better knowledge of these will help improve our understanding of the biological mechanisms of cell growth and development. In this study, a novel variant of porcine SIRT6 (SIRT6 V2) identified by reverse transcription-polymerase chain reaction and BLAST analysis showed a 124-bp deletion compared to wild-type SIRT6 mRNA (SIRT6 V1). Two recombinant plasmids overexpressing SIRT6 V1 and SIRT6 V2 were produced and their roles in the proliferation of porcine fetal fibroblasts (PFFs) were compared. Cells transfected with SIRT6 V1 proliferated significantly faster than those transfected with SIRT6 V2 (P<0.01), though both proliferated significantly faster than cells expressing an empty vector (P<0.01). These results might be caused by altered proportions of α helices and β sheets in the SIRT6 V2 structure relative to SIRT6 V1. These results indicate that overexpression of SIRT6 V1/V2 was involved in promoting PFF proliferation. Deletion of a 124-bp sequence attenuated the effects of SIRT6 on cell proliferation, possibly as a result of changes in the proportions of α helices and β sheets in the protein secondary structure.

Sirtuin 1-mediated Effects of Exercise and Resveratrol on Mitochondrial Biogenesis

The purpose of this study was to evaluate the role of sirtuin 1 (SirT1) in exercise- and resveratrol (RSV)-induced skeletal muscle mitochondrial biogenesis. Using muscle-specific SirT1-deficient (KO) mice and a cell culture model of differentiated myotubes, we compared the treatment of resveratrol, an activator of SirT1, with that of exercise in inducing mitochondrial biogenesis. These experiments demonstrated that SirT1 plays a modest role in maintaining basal mitochondrial content and a larger role in preserving mitochondrial function. Furthermore, voluntary exercise and RSV treatment induced mitochondrial biogenesis in a SirT1-independent manner. However, when RSV and exercise were combined, a SirT1-dependent synergistic effect was evident, leading to enhanced translocation of PGC-1α and SirT1 to the nucleus and stimulation of mitochondrial biogenesis. Thus, the magnitude of the effect of RSV on muscle mitochondrial biogenesis is reliant on SirT1, as well as the cellular environment, such as that produced by repeated bouts of exercise.