Genomic Copy-Number Loss Is Rescued by Self-Limiting Production of DNA Circles.

Copy-number changes generate phenotypic variability in health and disease. Whether organisms protect against copy-number changes is largely unknown. Here, we show that Saccharomyces cerevisiae monitors the copy number of its ribosomal DNA (rDNA) and rapidly responds to copy-number loss with the clonal amplification of extrachromosomal rDNA circles (ERCs) from chromosomal repeats. ERC formation is replicative, separable from repeat loss, and reaches a dynamic steady state that responds to the addition of exogenous rDNA copies. ERC levels are also modulated by RNAPI activity and diet, suggesting that rDNA copy number is calibrated against the cellular demand for rRNA. Last, we show that ERCs reinsert into the genome in a dosage-dependent manner, indicating that they provide a reservoir for ultimately increasing rDNA array length. Our results reveal a DNA-based mechanism for rapidly restoring copy number in response to catastrophic gene loss that shares fundamental features with unscheduled copy-number amplifications in cancer cells.

Yeast Crf1p is an activator with different roles in regulation of target genes.

Under stress conditions, ribosome biogenesis is downregulated. This process requires that expression of ribosomal RNA, ribosomal protein, and ribosome biogenesis genes be controlled in a coordinated fashion. The mechanistic Target of Rapamycin Complex 1 (mTORC1) participates in sensing unfavorable conditions to effect the requisite change in gene expression. In Saccharomyces cerevisiae, downregulation of ribosomal protein genes involves dissociation of the activator Ifh1p in a process that depends on Utp22p, a protein that also functions in pre-rRNA processing. Ifh1p has a paralog, Crf1p, which was implicated in communicating mTORC1 inhibition and hence was perceived as a repressor. We focus here on two ribosomal biogenesis genes, encoding Utp22p and the high mobility group protein Hmo1p, both of which are required for communication of mTORC1 inhibition to target genes. Crf1p functions as an activator on these genes as evidenced by reduced mRNA abundance and RNA polymerase II occupancy in a crf1Δ strain. Inhibition of mTORC1 has distinct effects on expression of HMO1 and UTP22; for example, on UTP22, but not on HMO1, the presence of Crf1p promotes the stable depletion of Ifh1p. Our data suggest that Crf1p functions as a weak activator, and that it may be required to prevent re-binding of Ifh1p to some gene promoters after mTORC1 inhibition in situations when Ifh1p is available. We propose that the inclusion of genes encoding proteins required for mTORC1-mediated downregulation of ribosomal protein genes in the same regulatory circuit as the ribosomal protein genes serves to optimize transcriptional responses during mTORC1 inhibition.

Interphase chromosome condensation in nutrient-starved conditions requires Cdc14 and Hmo1, but not condensin, in yeast.

When asynchronously growing cells suffer from nutrient depletion and inactivation of target of rapamycin complex 1 (TORC1) protein kinase, the rDNA (rRNA gene) region is condensed in budding yeast Saccharomyces cerevisiae, which is executed by condensin and Cdc14 protein phosphatase. However, it is unknown whether these mitotic factors can condense the rDNA region in nutrient-starved interphase cells. Here, we show that condensin is not involved in TORC1 inactivation-induced rDNA condensation in G1 cells. Instead, the high-mobility group protein Hmo1 drove this process. The histone deacetylase Rpd3 and Cdc14, which repress rRNA transcription, were both required for the interphase rDNA condensation. Furthermore, interphase rDNA condensation necessitated CLIP and cohibin that tether rDNA to inner nuclear membranes. Finally, we showed that Hmo1, CLIP, Rpd3, and Cdc14 were required for survival in nutrient-starved G1 cells. Thus, this study disclosed novel features of interphase chromosome condensation.

Control of RNA polymerase II-transcribed genes by direct binding of TOR kinase.

Under conditions of nutrient limitation and cellular stress, or by addition of rapamycin, the mechanistic target of rapamycin complex 1 (mTORC1) is inhibited. This results in downregulation of genes that encode rRNA and ribosomal proteins. While most of the mTORC1 functions that have been previously characterized at a mechanistic level take place in the cytoplasm, nuclear roles have also been reported, including direct association of TOR kinase with rRNA genes. This review highlights the recent observation that Saccharomyces cerevisiae Tor1p also binds directly to the RNA polymerase II-transcribed gene encoding Hmo1p, a protein that is involved in communicating mTORC1 activity to downstream targets. A reduction in HMO1 mRNA levels in response to DNA damage or addition of rapamycin requires Tor1p, suggesting a role for TOR kinase in control of gene activity by direct binding to target genes. Potential targets for chromatin-bound Tor1p are discussed and the possibility that Tor1p similarly contributes to control of other genes linked to ribosome biogenesis is considered.

HMGB proteins involved in TOR signaling as general regulators of cell growth by controlling ribosome biogenesis.

The number of ribosomes and their activity need to be highly regulated because their function is crucial for the cell. Ribosome biogenesis is necessary for cell growth and proliferation in accordance with nutrient availability and other external and intracellular signals. High-mobility group B (HMGB) proteins are conserved from yeasts to human and are decisive in cellular fate. These proteins play critical functions, from the maintenance of chromatin structure, DNA repair, or transcriptional regulation, to facilitation of ribosome biogenesis. They are also involved in cancer and other pathologies. In this review, we summarize evidence of how HMGB proteins contribute to ribosome-biogenesis control, with special emphasis on a common nexus to the target of rapamycin (TOR) pathway, a signaling cascade essential for cell growth and proliferation from yeast to human. Perspectives in this field are also discussed.

[HMGB Proteins as DNA Chaperones That Modulate Chromatin Activity].

HMGB proteins are involved in structural rearrangements caused by regulatory chromatin remodeling factors. Particular interest is attracted to a DNA chaperone mechanism, suggesting that the HMGB proteins introduce bends into the double helix, thus rendering DNA accessible to effector proteins and facilitating their activity. The review discusses the role that the HMBG proteins play in key intranuclear processes, including assembly of the preinitiation complex during transcription of ribosomal genes; transcription by RNA polymerases I, II, and III; recruitment of the SWI/SNF complex during transcription of nonribosomal genes; DNA repair; etc. The functions of the HMGB proteins are considered in detail with the examples of yeast HMO1 and NHP6. The two proteins possess unique features in adition to properties characteristic of the HMGB proteins. Thus, NHP6 stimulates a large-scale ATP-independent unwrapping of nucleosomal DNA by the FACT complex, while in its absence FACT stabilizes the nucleosome. HMO1 acts as an alternative linker histone. Both HMO1 and NHP6 are of applied interest primarly because they are homologs of human HMGB1, an important therapeutic target of anticancer and anti-inflammatory treatments.

Nucleosome remodeling by the SWI/SNF complex is enhanced by yeast high mobility group box (HMGB) proteins.

The regulation of gene expression at the level of transcription involves the concerted action of several proteins and protein complexes committed to dynamically alter the surrounding chromatin environment of a gene being activated or repressed. ATP-dependent chromatin remodeling complexes are key factors in chromatin remodeling, and the SWI/SNF complex is the founding member. While many studies have linked the action of these complexes to specific transcriptional regulation of a large number of genes and much is known about their catalytic activity, less is known about the nuclear elements that can enhance or modulate their activity. A number of studies have found that certain High Mobility Group (HMG) proteins are able to stimulate ATP-dependent chromatin remodeling activity, but their influence on the different biochemical outcomes of this activity is still unknown. In this work we studied the influence of the yeast Nhp6A, Nhp6B and Hmo1 proteins (HMGB family members) on different biochemical outcomes of yeast SWI/SNF remodeling activity. We found that all these HMG proteins stimulate the sliding activity of ySWI/SNF, while transient exposure of nucleosomal DNA and octamer transfer catalyzed by this complex are only stimulated by Hmo1. Consistently, only Hmo1 stimulates SWI/SNF binding to the nucleosome. Additionally, the sliding activity of another chromatin remodeling complex, ISW1a, is only stimulated by Hmo1. Further analyses show that these differential stimulatory effects of Hmo1 are dependent on the presence of its C-terminal tail, which contains a stretch of acidic and basic residues.

Complexes of HMO1 with DNA: Structure and Affinity.

Saccharomyces cerevisiae HMO1 is an architectural nuclear DNA-binding protein that stimulates the activity of some remodelers and regulates the transcription of ribosomal protein genes, often binding to a DNA motif called IFHL. However, the molecular mechanism dictating this sequence specificity is unclear. Our circular dichroism spectroscopy studies show that the HMO1:DNA complex forms without noticeable changes in the structure of DNA and HMO1. Molecular modeling/molecular dynamics studies of the DNA complex with HMO1 Box B reveal two extended sites at the N-termini of helices I and II of Box B that are involved in the formation of the complex and stabilize the DNA bend induced by intercalation of the F114 side chain between base pairs. A comparison of the affinities of HMO1 for 24 bp DNA fragments containing either randomized or IFHL sequences reveals a twofold increase in the stability of the complex in the latter case, which may explain the selectivity in the recognition of the IFHL-containing promoter regions.

Hmo1: A versatile member of the high mobility group box family of chromosomal architecture proteins.

Eukaryotic chromatin consisting of nucleosomes connected by linker DNA is organized into higher order structures, which is facilitated by linker histone H1. Formation of chromatin compacts and protects the genome, but also hinders DNA transactions. Cells have evolved mechanisms to modify/remodel chromatin resulting in chromatin states suitable for genome functions. The high mobility group box (HMGB) proteins are non-histone chromatin architectural factors characterized by one or more HMGB motifs that bind DNA in a sequence nonspecific fashion. They play a major role in chromatin dynamics. The Saccharomyces cerevisiae (yeast hereafter) HMGB protein Hmo1 contains two HMGB motifs. However, unlike a canonical HMGB protein that has an acidic C-terminus, Hmo1 ends with a lysine rich, basic, C-terminus, resembling linker histone H1. Hmo1 exhibits characteristics of both HMGB proteins and linker histones in its multiple functions. For instance, Hmo1 promotes transcription by RNA polymerases I and II like canonical HMGB proteins but makes chromatin more compact/stable like linker histones. Recent studies have demonstrated that Hmo1 destabilizes/disrupts nucleosome similarly as other HMGB proteins in vitro and acts to maintain a common topological architecture of genes in yeast genome. This minireview reviews the functions of Hmo1 and the underlying mechanisms, highlighting recent discoveries.

Single-molecule study reveals Hmo1, not Hho1, promotes chromatin assembly in budding yeast.

Linker histone H1 plays a crucial role in various biological processes, including nucleosome stabilization, high-order chromatin structure organization, gene expression, and epigenetic regulation in eukaryotic cells. Unlike higher eukaryotes, little about the linker histone in Saccharomyces cerevisiae is known. Hho1 and Hmo1 are two long-standing controversial histone H1 candidates in budding yeast. In this study, we directly observed at the single-molecule level that Hmo1, but not Hho1, is involved in chromatin assembly in the yeast nucleoplasmic extracts (YNPE), which can replicate the physiological condition of the yeast nucleus. The presence of Hmo1 facilitates the assembly of nucleosomes on DNA in YNPE, as revealed by single-molecule force spectroscopy. Further single-molecule analysis showed that the lysine-rich C-terminal domain (CTD) of Hmo1 is essential for the function of chromatin compaction, while the second globular domain at the C-terminus of Hho1 impairs its ability. In addition, Hmo1, but not Hho1, forms condensates with double-stranded DNA via reversible phase separation. The phosphorylation fluctuation of Hmo1 coincides with metazoan H1 during the cell cycle. Our data suggest that Hmo1, but not Hho1, possesses some functionality similar to that of linker histone in Saccharomyces cerevisiae, even though some properties of Hmo1 differ from those of a canonical linker histone H1. Our study provides clues for the linker histone H1 in budding yeast and provides insights into the evolution and diversity of histone H1 across eukaryotes. IMPORTANCE There has been a long-standing debate regarding the identity of linker histone H1 in budding yeast. To address this issue, we utilized YNPE, which accurately replicate the physiological conditions in yeast nuclei, in combination with total internal reflection fluorescence microscopy and magnetic tweezers. Our findings demonstrated that Hmo1, rather than Hho1, is responsible for chromatin assembly in budding yeast. Additionally, we found that Hmo1 shares certain characteristics with histone H1, including phase separation and phosphorylation fluctuations throughout the cell cycle. Furthermore, we discovered that the lysine-rich domain of Hho1 is buried by its second globular domain at the C-terminus, resulting in the loss of function that is similar to histone H1. Our study provides compelling evidence to suggest that Hmo1 shares linker histone H1 function in budding yeast and contributes to our understanding of the evolution of linker histone H1 across eukaryotes.

The linker histone Hho1 modulates the activity of ATP-dependent chromatin remodeling complexes.

Diverse factors play roles in chromatin dynamics, including linker proteins. Among them are high mobility group (HMG) box family proteins and linker histones. In the yeast Saccharomyces cerevisiae, Hmo1 has been identified as an HMG-box protein. This protein displays properties that are in agreement with this allocation. However, a number of studies have postulated that Hmo1 functions as a linker histone in yeast. On the other hand, when discovered, the Hho1 protein was identified as a linker histone. While multiple studies support this classification, some findings point to characteristics of Hho1 that are dissimilar to those commonly assigned to linker histones. In order to better understand the roles played by Hmo1 and Hho1 in chromatin dynamics and transcriptional regulation, we performed several analyses directly comparing these two proteins. Our analyses of genome-wide binding profiles support the belonging of Hmo1 to the HMGB family and Hho1 to the linker histones family. Interestingly, by performing protein-protein interaction analyses we found that both Hmo1 and Hho1 display physical interaction with the ATP-dependent chromatin remodeling complexes RSC, ISW1a and SWI/SNF. Moreover, by carrying out nucleosome remodeling assays, we found that both proteins stimulate the activity of the ISW1a complex. However, in the case of RSC, Hmo1 and Hho1 displayed differential properties, with Hho1 mainly showing an inhibitory effect. Our results are in agreement with the opposite roles played by RSC and ISW1a in chromatin dynamics and transcriptional regulation, and expand the view for the roles played by Hho1 and linker histones.

Hmo1 Protein Affects the Nucleosome Structure and Supports the Nucleosome Reorganization Activity of Yeast FACT.

Yeast Hmo1 is a high mobility group B (HMGB) protein that participates in the transcription of ribosomal protein genes and rDNA, and also stimulates the activities of some ATP-dependent remodelers. Hmo1 binds both DNA and nucleosomes and has been proposed to be a functional yeast analog of mammalian linker histones. We used EMSA and single particle Förster resonance energy transfer (spFRET) microscopy to characterize the effects of Hmo1 on nucleosomes alone and with the histone chaperone FACT. Hmo1 induced a significant increase in the distance between the DNA gyres across the nucleosomal core, and also caused the separation of linker segments. This was opposite to the effect of the linker histone H1, which enhanced the proximity of linkers. Similar to Nhp6, another HMGB factor, Hmo1, was able to support large-scale, ATP-independent, reversible unfolding of nucleosomes by FACT in the spFRET assay and partially support FACT function in vivo. However, unlike Hmo1, Nhp6 alone does not affect nucleosome structure. These results suggest physiological roles for Hmo1 that are distinct from Nhp6 and possibly from other HMGB factors and linker histones, such as H1.

Establishment and Maintenance of Open Ribosomal RNA Gene Chromatin States in Eukaryotes.

In growing eukaryotic cells, nuclear ribosomal (r)RNA synthesis by RNA polymerase (RNAP) I accounts for the vast majority of cellular transcription. This high output is achieved by the presence of multiple copies of rRNA genes in eukaryotic genomes transcribed at a high rate. In contrast to most of the other transcribed genomic loci, actively transcribed rRNA genes are largely devoid of nucleosomes adapting a characteristic "open" chromatin state, whereas a significant fraction of rRNA genes resides in a transcriptionally inactive nucleosomal "closed" chromatin state. Here, we review our current knowledge about the nature of open rRNA gene chromatin and discuss how this state may be established.

Yeast Crf1p: An activator in need is an activator indeed.

Ribosome biogenesis is an energetically costly process, and tight regulation is required for stoichiometric balance between components. This requires coordination of RNA polymerases I, II, and III. Lack of nutrients or the presence of stress leads to downregulation of ribosome biogenesis, a process for which mechanistic target of rapamycin complex I (mTORC1) is key. mTORC1 activity is communicated by means of specific transcription factors, and in yeast, which is a primary model system in which transcriptional coordination has been delineated, transcription factors involved in regulation of ribosomal protein genes include Fhl1p and its cofactors, Ifh1p and Crf1p. Ifh1p is an activator, whereas Crf1p has been implicated in maintaining the repressed state upon mTORC1 inhibition. Computational analyses of evolutionary relationships have indicated that Ifh1p and Crf1p descend from a common ancestor. Here, we discuss recent evidence, which suggests that Crf1p also functions as an activator. We propose a model that consolidates available experimental evidence, which posits that Crf1p functions as an alternate activator to prevent the stronger activator Ifh1p from re-binding gene promoters upon mTORC1 inhibition. The correlation between retention of Crf1p in related yeast strains and duplication of ribosomal protein genes suggests that this backup activation may be important to ensure gene expression when Ifh1p is limiting. With ribosome biogenesis as a hallmark of cell growth, failure to control assembly of ribosomal components leads to several human pathologies. A comprehensive understanding of mechanisms underlying this process is therefore of the essence.

Specialization of the chromatin remodeler RSC to mobilize partially-unwrapped nucleosomes.

SWI/SNF-family chromatin remodeling complexes, such as S. cerevisiae RSC, slide and eject nucleosomes to regulate transcription. Within nucleosomes, stiff DNA sequences confer spontaneous partial unwrapping, prompting whether and how SWI/SNF-family remodelers are specialized to remodel partially-unwrapped nucleosomes. RSC1 and RSC2 are orthologs of mammalian PBRM1 (polybromo) which define two separate RSC sub-complexes. Remarkably, in vitro the Rsc1-containing complex remodels partially-unwrapped nucleosomes much better than does the Rsc2-containing complex. Moreover, a rsc1Δ mutation, but not rsc2Δ, is lethal with histone mutations that confer partial unwrapping. Rsc1/2 isoforms both cooperate with the DNA-binding proteins Rsc3/30 and the HMG protein, Hmo1, to remodel partially-unwrapped nucleosomes, but show differential reliance on these factors. Notably, genetic impairment of these factors strongly reduces the expression of genes with wide nucleosome-deficient regions (e.g., ribosomal protein genes), known to harbor partially-unwrapped nucleosomes. Taken together, Rsc1/2 isoforms are specialized through composition and interactions to manage and remodel partially-unwrapped nucleosomes.

rDNA Condensation Promotes rDNA Separation from Nucleolar Proteins Degraded for Nucleophagy after TORC1 Inactivation.

Nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase induce nucleophagy preferentially degrading only nucleolar components in budding yeast. Nucleolar proteins are relocated to sites proximal to the nucleus-vacuole junction (NVJ), where micronucleophagy occurs, whereas rDNA, which is embedded in the nucleolus under normal conditions, moves to NVJ-distal regions, causing rDNA dissociation from nucleolar proteins after TORC1 inactivation. This repositioning is mediated via chromosome linkage INM protein (CLIP)-cohibin complexes that tether rDNA to the inner nuclear membrane. Here, we show that TORC1 inactivation-induced rDNA condensation promotes the repositioning of rDNA and nucleolar proteins. Defects in condensin, Rpd3-Sin3 histone deacetylase (HDAC), and high-mobility group protein 1 (Hmo1), which are involved in TORC1 inactivation-induced rDNA condensation, compromised the repositioning and nucleophagic degradation of nucleolar proteins, although rDNA still escaped from nucleophagic degradation in these mutants. We propose a model in which rDNA condensation after TORC1 inactivation generates a motive force for the repositioning of rDNA and nucleolar proteins.

Role of Nhp6 and Hmo1 in SWI/SNF occupancy and nucleosome landscape at gene regulatory regions.

Diverse chromatin modifiers are involved in regulation of gene expression at the level of transcriptional regulation. Among these modifiers are ATP-dependent chromatin remodelers, where the SWI/SNF complex is the founding member. It has been observed that High Mobility Group (HMG) proteins can influence the activity of a number of these chromatin remodelers. In this context, we have previously demonstrated that the yeast HMG proteins Nhp6 and Hmo1 can stimulate SWI/SNF activity. Here, we studied the genome-wide binding patterns of Nhp6, Hmo1 and the SWI/SNF complex, finding that most of gene promoters presenting high occupancy of this complex also display high enrichment of these HMG proteins. Using deletion mutant strains we demonstrate that binding of SWI/SNF is significantly reduced at numerous genomic locations by deletion of NHP6 and/or deletion of HMO1. Moreover, alterations in the nucleosome landscape take place at gene promoters undergoing reduced SWI/SNF binding. Additional analyses show that these effects also correlate with alterations in transcriptional activity. Our results suggest that, besides the ability to stimulate SWI/SNF activity, these HMG proteins are able to assist the loading of this complex onto gene regulatory regions.

Coordination of Ribosomal Protein and Ribosomal RNA Gene Expression in Response to TOR Signaling.

Cells grow in response to nutrients or growth factors, whose presence is detected and communicated by elaborate signaling pathways. Protein kinases play crucial roles in processes such as cell cycle progression and gene expression, and misregulation of such pathways has been correlated with various diseased states. Signals intended to promote cell growth converge on ribosome biogenesis, as the ability to produce cellular proteins is intimately tied to cell growth. Part of the response to growth signals is therefore the coordinate expression of genes encoding ribosomal RNA (rRNA) and ribosomal proteins (RP). A key player in regulating cell growth is the Target of Rapamycin (TOR) kinase, one of the gatekeepers that prevent cell cycle progression from G1 to S under conditions of nutritional stress. TOR is structurally and functionally conserved in all eukaryotes. Under favorable growth conditions, TOR is active and cells maintain a robust rate of ribosome biogenesis, translation initiation and nutrient import. Under stress conditions, TOR signaling is suppressed, leading to cell cycle arrest, while the failure of TOR to respond appropriately to environmental or nutritional signals leads to uncontrolled cell growth. Emerging evidence from Saccharomyces cerevisiae indicates that High Mobility Group (HMGB) proteins, non-sequence-specific chromosomal proteins, participate in mediating responses to growth signals. As HMGB proteins are distinguished by their ability to alter DNA topology, they frequently function in the assembly of higher-order nucleoprotein complexes. We review here recent evidence, which suggests that HMGB proteins may function to coordinate TOR-dependent regulation of rRNA and RP gene expression.