Tho2-mediated escort of Nrd1 regulates the expression of aging-related genes.

The relationship between aging and RNA biogenesis and trafficking is attracting growing interest, yet the precise mechanisms are unknown. The THO complex is crucial for mRNA cotranscriptional maturation and export. Herein, we report that the THO complex is closely linked to the regulation of lifespan. Deficiencies in Hpr1 and Tho2, components of the THO complex, reduced replicative lifespan (RLS) and are linked to a novel Sir2-independent RLS control pathway. Although transcript sequestration in hpr1Δ or tho2Δ mutants was countered by exosome component Rrp6, loss of this failed to mitigate RLS defects in hpr1Δ. However, RLS impairment in hpr1Δ or tho2Δ was counteracted by the additional expression of Nrd1-specific mutants that interacted with Rrp6. This effect relied on the interaction of Nrd1, a transcriptional regulator of aging-related genes, including ribosome biogenesis or RNA metabolism genes, with RNA polymerase II. Nrd1 overexpression reduced RLS in a Tho2-dependent pathway. Intriguingly, Tho2 deletion mirrored Nrd1 overexpression effects by inducing arbitrary Nrd1 chromatin binding. Furthermore, our genome-wide ChIP-seq analysis revealed an increase in the recruitment of Nrd1 to translation-associated genes, known to be related to aging, upon Tho2 loss. Taken together, these findings underscore the importance of Tho2-mediated Nrd1 escorting in the regulation of lifespan pathway through transcriptional regulation of aging-related genes.

Sus1 maintains a normal lifespan through regulation of TREX-2 complex-mediated mRNA export.

Eukaryotic gene expression requires multiple cellular events, including transcription and RNA processing and transport. Sus1, a common subunit in both the Spt-Ada-Gcn5 acetyltransferase (SAGA) and transcription and export complex-2 (TREX-2) complexes, is a key factor in coupling transcription activation to mRNA nuclear export. Here, we report that the SAGA DUB module and TREX-2 distinctly regulate yeast replicative lifespan in a Sir2-dependent and -independent manner, respectively. The growth and lifespan impaired by SUS1 loss depend on TREX-2 but not on the SAGA DUB module. Notably, an increased dose of the mRNA export factors Mex67 and Dbp5 rescues the growth defect, shortened lifespan, and nuclear accumulation of poly(A)+ RNA in sus1Δ cells, suggesting that boosting the mRNA export process restores the mRNA transport defect and the growth and lifespan damage in sus1Δ cells. Moreover, Sus1 is required for the proper association of Mex67 and Dbp5 with the nuclear rim. Together, these data indicate that Sus1 links transcription and mRNA nuclear export to the lifespan control pathway, suggesting that prevention of an abnormal accumulation of nuclear RNA is necessary for maintenance of a normal lifespan.

Nuclear mRNA Export and Aging.

The relationship between transcription and aging is one that has been studied intensively and experimentally with diverse attempts. However, the impact of the nuclear mRNA export on the aging process following its transcription is still poorly understood, although the nuclear events after transcription are coupled closely with the transcription pathway because the essential factors required for mRNA transport, namely TREX, TREX-2, and nuclear pore complex (NPC), physically and functionally interact with various transcription factors, including the activator/repressor and pre-mRNA processing factors. Dysregulation of the mediating factors for mRNA export from the nucleus generally leads to the aberrant accumulation of nuclear mRNA and further impairment in the vegetative growth and normal lifespan and the pathogenesis of neurodegenerative diseases. The optimal stoichiometry and density of NPC are destroyed during the process of cellular aging, and their damage triggers a defect of function in the nuclear permeability barrier. This review describes recent findings regarding the role of the nuclear mRNA export in cellular aging and age-related neurodegenerative disorders.

Epigenetic Targeting of Histone Deacetylases in Diagnostics and Treatment of Depression.

Depression is a highly prevalent, disabling, and often chronic illness that places substantial burdens on patients, families, healthcare systems, and the economy. A substantial minority of patients are unresponsive to current therapies, so there is an urgent need to develop more broadly effective, accessible, and tolerable therapies. Pharmacological regulation of histone acetylation level has been investigated as one potential clinical strategy. Histone acetylation status is considered a potential diagnostic biomarker for depression, while inhibitors of histone deacetylases (HDACs) have garnered interest as novel therapeutics. This review describes recent advances in our knowledge of histone acetylation status in depression and the therapeutic potential of HDAC inhibitors.

The Spt7 subunit of the SAGA complex is required for the regulation of lifespan in both dividing and nondividing yeast cells.

Spt7 belongs to the suppressor of Ty (SPT) module of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex and is known as the yeast ortholog of human STAF65γ. Spt7 lacks intrinsic enzymatic activity but is responsible for the integrity and proper assembly of the SAGA complex. Here, we determined the role of the SAGA Spt7 subunit in cellular aging. We found that Spt7 was indispensable for a normal lifespan in both dividing and nondividing yeast cells. In the quiescent state of cells, Spt7 was required for the control of overall mRNA levels. In mitotically active cells, deletion of the SPT module had little effect on the recombination rate within heterochromatic ribosomal DNA (rDNA) loci, but loss of Spt7 profoundly elevated the plasmid-based DNA recombination frequency. Consistently, loss of Spt7 increased spontaneous Rad52 foci by approximately two-fold upon entry into S phase. These results provide evidence that Spt7 contributes to the regulation of the normal replicative lifespan (RLS) and chronological lifespan (CLS), possibly by controlling the DNA recombination rate and overall mRNA expression. We propose that the regulation of SAGA complex integrity by Spt7 might be involved in the conserved regulatory pathway for lifespan regulation in eukaryotes.

SUMO and cellular adaptive mechanisms.

The ubiquitin family member SUMO is a covalent regulator of proteins that functions in response to various stresses, and defects in SUMO-protein conjugation or deconjugation have been implicated in multiple diseases. The loss of the Ulp2 SUMO protease, which reverses SUMO-protein modifications, in the model eukaryote Saccharomyces cerevisiae is severely detrimental to cell fitness and has emerged as a useful model for studying how cells adapt to SUMO system dysfunction. Both short-term and long-term adaptive mechanisms are triggered depending on the length of time cells spend without this SUMO chain-cleaving enzyme. Such short-term adaptations include a highly specific multichromosome aneuploidy and large changes in ribosomal gene transcription. While aneuploid ulp2Δ cells survive, they suffer severe defects in growth and stress resistance. Over many generations, euploidy is restored, transcriptional programs are adjusted, and specific genetic changes that compensate for the loss of the SUMO protease are observed. These long-term adapted cells grow at normal rates with no detectable defects in stress resistance. In this review, we examine the connections between SUMO and cellular adaptive mechanisms more broadly.

Symmetric dimethylation on histone H4R3 associates with histone deacetylation to maintain properly polarized cell growth.

Yeast Hsl7 is recognized as a homolog of human arginine methyltransferase 5 (PRMT5) and shows type II PRMT activity by forming symmetric dimethylarginine residues on histones. Previously, we reported that Hsl7 is responsible for in vivo symmetric dimethylation on histone H4 arginine 3 (H4R3me2s) in a transcriptionally repressed state, possibly in association with histone deacetylation by Rpd3. Here, we investigated the function of Hsl7 during cell cycle progression. We found that the accumulation of Hsl7-mediated H4R3me2s is maintained by the histone deacetylase Rpd3 during transcriptional repression and that the low level of H4R3me2s is required for proper asymmetric cell growth during cell division. Our results suggest that the hypoacetylated state of histones is connected to the function of Hsl7 in regulating properly polarized cell growth during cell division and provide new insight into the epigenetic modifications that are important for cell cycle morphogenesis checkpoint control based on the repressive histone crosstalk between symmetric arginine methylation of H4 and histone deacetylation.

Multiple roles of CTDK-I throughout the cell.

The heterotrimeric carboxy-terminal domain kinase I (CTDK-I) in yeast is a cyclin-dependent kinase complex that is evolutionally conserved throughout eukaryotes and phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (RNApII) on the second-position serine (Ser2) residue of YSPTSPS heptapeptide repeats. CTDK-I plays indispensable roles in transcription elongation and transcription-coupled processing, such as the 3'-end processing of nascent mRNA transcripts. However, recent studies have revealed additional roles of CTDK-I beyond its primary effect on transcription by RNApII. Here, we describe recent advances in the regulation of genomic stability and rDNA integrity by CTDK-I and highlight the previously underappreciated cellular roles of CTDK-I in rRNA synthesis by RNA polymerase I and translational initiation and elongation. These multiple roles of CTDK-I throughout the cell expand our understanding of how this complex functions to coordinate diverse cellular processes through gene expression and how the human orthologue exerts its roles in diseased states such as tumorigenesis.

Yeast symmetric arginine methyltransferase Hsl7 has a repressive role in transcription.

Protein arginine methylation, an evolutionarily conserved post-translational modification, serves critical cellular functions by transferring a methyl group to a variety of substrates, including histones and some transcription factors. In budding yeast, Hsl7 (histone synthetic lethal 7) displays type II PRMT (protein arginine methyltransferase) activity by generating symmetric dimethylarginine residues on histone H2A in vitro. However, identification of the in vivo substrate of Hsl7 and how it contributes to important cellular processes remain largely unexplored. In the present study, we show that Hsl7 has a repressive role in transcription. We found that Hsl7 is responsible for in vivo symmetric dimethylation of histone H4 arginine 3 (H4R3me2s) in a transcriptionally repressed state. Tandem affinity purification further demonstrated that Hsl7 physically interacts with histone deacetylase Rpd3, and both similarly repress transcription. Our results suggest that H4R3me2s generation by the type II PRMT Hsl7 is required for transcriptional repression, possibly in cooperation with histone deacetylation by Rpd3.

The Epigenetic Pathways to Ribosomal DNA Silencing.

Heterochromatin is the transcriptionally repressed portion of eukaryotic chromatin that maintains a condensed appearance throughout the cell cycle. At sites of ribosomal DNA (rDNA) heterochromatin, epigenetic states contribute to gene silencing and genome stability, which are required for proper chromosome segregation and a normal life span. Here, we focus on recent advances in the epigenetic regulation of rDNA silencing in Saccharomyces cerevisiae and in mammals, including regulation by several histone modifications and several protein components associated with the inner nuclear membrane within the nucleolus. Finally, we discuss the perturbations of rDNA epigenetic pathways in regulating cellular aging and in causing various types of diseases.

Modifications of RNA polymerase II CTD: Connections to the histone code and cellular function.

At the onset of transcription, many protein machineries interpret the cellular signals that regulate gene expression. These complex signals are mostly transmitted to the indispensable primary proteins involved in transcription, RNA polymerase II (RNAPII) and histones. RNAPII and histones are so well coordinated in this cellular function that each cellular signal is precisely allocated to specific machinery depending on the stage of transcription. The carboxy-terminal domain (CTD) of RNAPII in eukaryotes undergoes extensive posttranslational modification, called the 'CTD code', that is indispensable for coupling transcription with many cellular processes, including mRNA processing. The posttranslational modification of histones, known as the 'histone code', is also critical for gene transcription through the reversible and dynamic remodeling of chromatin structure. Notably, the histone code is closely linked with the CTD code, and their combinatorial effects enable the delicate regulation of gene transcription. This review elucidates recent findings regarding the CTD modifications of RNAPII and their coordination with the histone code, providing integrative pathways for the fine-tuned regulation of gene expression and cellular function.

Loss of the Set2 histone methyltransferase increases cellular lifespan in yeast cells.

The post-translational modification of histones has been implicated in the regulation of cellular lifespan. Previously, we reported that cellular aging is associated with increased ubiquitylation of histone H2B and methylation of histone H3 at lysines 4 and 79 in yeast telomeric heterochromatin. Here, we show the antagonistic role of Set2 methyltransferase, which is specific for histone H3 at lysine 36, in regulating telomeric silencing and cellular lifespan. We observed that an intermediate state of chromatin, namely, unstable ON telomeres, exists when a gene is switched on near telomeres. This unstable state of chromatin is temporally maintained in a transcription-dependent manner and is preferentially restored to its original heterochromatic state, namely, OFF telomeres. We found that Set2 suppresses the restoration of unstable ON telomeres to the stable OFF state and promotes cellular aging. Our results suggest that the accumulation of unstable ON telomeres maintained by Set2 is one of the features of aged cells.

The RNA polymerase II Rpb4/7 subcomplex regulates cellular lifespan through an mRNA decay process.

In budding yeast, a highly conserved heterodimeric protein complex that is composed of the Rpb4 and Rpb7 proteins within RNA polymerase II shuttles between the nucleus and cytoplasm where it coordinates various steps of gene expression by associating with mRNAs. Although distinct stages of gene expression potentially contribute to the regulation of cellular lifespan, little is known about the underlying mechanisms. Here, we addressed the role of the dissociable Rpb4/7 heterodimeric protein complex in the regulation of replicative lifespan during various stages of gene expression in the yeast Saccharomyces cerevisiae. We observed that the loss of Rpb4 resulted in a shortened lifespan. In contrast, we found that defects in the dissociation of Rpb4/7 from the RNA polymerase core complex and in translation initiation steps affected by Rpb4/7 did not impact lifespan. Tandem affinity purification experiments demonstrated that Rpb7 physically associates with Tpk2 and Pat1, which are both implicated in mRNA degradation. Consistent with this data, the loss of the mRNA decay regulators Pat1 and Dhh1 reduced the cellular lifespan. In summary, our findings further reinforce the pivotal role of Rpb4/7 in the coordination of distinct steps of gene expression and suggest that among the many stages of gene expression, mRNA decay is a critical process that is required for normal replicative lifespan.

Cellular aging is associated with increased ubiquitylation of histone H2B in yeast telomeric heterochromatin.

Epigenetic changes in chromatin state are associated with aging. Notably, two histone modifications have recently been implicated in lifespan regulation, namely acetylation at H4 lysine 16 in yeast and methylation at H3 lysine 4 (H3K4) in nematodes. However, less is known about other histone modifications. Here, we report that cellular aging is associated with increased ubiquitylation of histone H2B in yeast telomeric heterochromatin. An increase in ubiquitylation at histone H2B lysine 123 and methylations at both H3K4 and H3 lysine 79 (H3K79) was observed at the telomere-proximal regions of replicatively aged cells, coincident with decreased Sir2 abundance. Moreover, deficiencies in the H2B ubiquitylase complex Rad6/Bre1 as well as the deubiquitylase Ubp10 reduced the lifespan by altering both H3K4 and H3K79 methylation and Sir2 recruitment. Thus, these results show that low levels of H2B ubiquitylation are a prerequisite for a normal lifespan and the trans-tail regulation of histone modifications regulates age-associated Sir2 recruitment through telomeric silencing.

Role of yeast JmjC-domain containing histone demethylases in actively transcribed regions.

In budding yeast, there are five JmjC domain-containing proteins, Jhd1, Jhd2, Rph1, Ecm5, and Gis1, which have been suggested to directly remove histone lysine methylation via a hydroxylation reaction. Of these demethylases, the ability of Jhd1 or Rph1 to demethylate histone H3 as a substrate has been identified in vivo. However, the overall roles of endogenous JmjC demethylases in the demethylation of histones encompassed by genes that are constitutively transcribed or their specificities towards histone H3 lysine modification at mono-, di-, or trimethylation states are still unclear. Using chromatin immunoprecipitation with nine specific antibodies directed against mono-, di-, or trimethylated histone H3 at lysines 4, 36, or 79, we show the whole patterns of histone H3 lysine methylation and the net changes in methylations that are caused by the deletion of each of the five JmjC demethylases in actively transcribed regions. Our results show that of the JmjC-containing proteins, Rph1 is the demethylase that is specific for histone H3K36 trimethylation during transcription elongation in vivo, and the abilities of other endogenous JmjC demethylasesto demethylate histone H3 are weak toward histone H3in actively transcribed regions.

IMP dehydrogenase is recruited to the transcription complex through serine 2 phosphorylation of RNA polymerase II.

IMP dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo synthesis of guanine, namely the oxidation of IMP to XMP with a concomitant reduction of NAD+. In Saccharomyces cerevisiae, a family of four closely-related genes, IMD1, IMD2 (also known as PUR5), IMD3, and IMD4, encodes the putative IMPDH. Although IMPDH synthesizes guanine in the cytoplasm, it has also been found in the nucleus, where it associates with nucleic acids in human cells. Here, we further show that IMPDH is recruited to actively transcribed region of genes. A synthetic lethal screen using a deletion strain of Ctk1 kinase, a yeast homolog of mammalian Cdk9/P-TEFb that phosphorylates serine 2 within the RNA polymerase II (RNApII) C-terminal domain (CTD), identified that Imd2 genetically interacts with Ctk1. Consistent with this association, IMPDHs were recruited to elongating RNApII only when serine 2 of the CTD was phosphorylated by Ctk1. Loss of Imd2 had little effect on the association of most elongation factors with RNApII. However, in cells lacking Imd2 or all the essential IMPDHs in the presence of minimal guanine, a defect in the association of Ctk1 with the promoter region was seen. Taken together, our results show that IMPDH is recruited to transcription complex through serine 2 phosphorylation of RNApII CTD and suggest that it may play a role in initiating transcriptional regulation.

A Bre1-associated protein, large 1 (Lge1), promotes H2B ubiquitylation during the early stages of transcription elongation.

Transcription activation has been proposed to require both ubiquitylation and deubiquitylation of histone H2B. Here, we show that Lge1 (Large 1) is found in a complex containing Rad6.Bre1 and that it controls the recruitment of Bre1, a ubiquitin ligase, and Ubp8, a deubiquitylase, to promote ubiquitylation during the early steps in elongation. Chromatin immunoprecipitation experiments showed that Lge1 associates with promoter and coding regions of actively transcribed genes in a transcription-dependent manner. Disruption of Lge1 abolished ubiquitylation of histone H2B on lysine 123 and H3 methylation on lysines 4 and 79 and resulted in significant sensitivity to 6-azauracil and mycophenolic acid. In particular, in Lge1-deficient cells, Bre1 recruitment was attenuated, whereas recruitment of Ubp8 was facilitated. These alterations were coincident with changes in the interaction between Bre1.Ubp8 and RNA polymerase II phosphorylated at serine 5 of the C-terminal domain. We propose that Lge1 has a novel function in disrupting the balance between the recruitment of Bre1 and Ubp8, thus promoting transcription elongation.

A novel gamma-lactam-based histone deacetylase inhibitor potently inhibits the growth of human breast and renal cancer cells.

We evaluated the novel gamma-lactam-based analogue, KBH-A145, for its anticancer activities. KBH-A145 markedly inhibited histone deacetylase (HDAC) activity in vitro and in vivo to an extent comparable to suberoyl-anilide hydroxamic acid (SAHA). The proliferation of various types of cancers was significantly suppressed by KBH-A145, among which MDA-MB-231 and MCF, human breast cancer cells and ACHN human renal cancer cells, were most sensitive. This was accompanied by induction of p21(WAF1/Cip1) through compromised recruitment of HDAC1, which leads to hyperacetylation of its promoter region and thus arrested both cells in the G(2)/M phase. Interestingly, this compound induced apoptosis of MDA-MB-231 cells, but not ACHN cells, through cleavage of poly(ADP-ribose) polymerase (PARP). Taken together, these results show that this novel gamma-lactam-based HDAC inhibitor potently inhibits the growth of human breast and renal cancer cells. Thus KBH-A145 is a potential therapeutic agent for the treatment of these types of cancer.

Rapamycin down-regulates inducible nitric oxide synthase by inducing proteasomal degradation.

We investigated the effect of rapamycin, a specific inhibitor of the mammalian serine/threonine kinase, mammalian target of rapamycin (mTOR), on the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Pretreatment of cells with rapamycin significantly inhibited LPS-induced nitrite production and the expression of iNOS protein in a dose-dependent manner. However, LPS-induced mRNA expression of iNOS and its concomitant activation of nuclear factor (NF)-kappaB remained unchanged by rapamycin. Intriguingly, LPS-induced nitrite production and iNOS protein expression were partially blocked at nanomolar concentrations of rapamycin, whereas phosphorylation of both p70 S6 kinase and 4E-BP1 was completely abolished. The suppression of LPS-induced iNOS expression by rapamycin was reversed by the protease inhibitor lactacystin. Furthermore, rapamycin treatment stimulated 20S proteasome activity, which was slightly elevated by LPS. Taken together, our findings strongly suggest that rapamycin down-regulates LPS-induced iNOS protein expression via proteasomal activation, as well as through inhibition of the mTOR signaling pathway.

Ctk1 promotes dissociation of basal transcription factors from elongating RNA polymerase II.

As RNA polymerase II (RNApII) transitions from initiation to elongation, Mediator and the basal transcription factors TFIID, TFIIA, TFIIH, and TFIIE remain at the promoter as part of a scaffold complex, whereas TFIIB and TFIIF dissociate. The yeast Ctk1 kinase associates with elongation complexes and phosphorylates serine 2 in the YSPTSPS repeats of the Rpb1 C-terminal domain, a modification that couples transcription to mRNA 3'-end processing. The higher eukaryotic kinase Cdk9 not only performs a similar function, but also functions at the 5'-end of genes in the transition from initiation to elongation. In strains lacking Ctk1, many basal transcription factors cross-link throughout transcribed regions, apparently remaining associated with RNApII until it terminates. Consistent with this observation, preinitiation complexes formed on immobilized templates with transcription extracts lacking Ctk1 leave lower levels of the scaffold complex behind after escape. Taken together, these results suggest that Ctk1 is necessary for the release of RNApII from basal transcription factors. Interestingly, this function of Ctk1 is independent of its kinase activity, suggesting a structural function of the protein.