Daniel Gottschling

• PhD
• Principal Investigator at Calico Labs

Amino acid homeostasis is critical for many cellular processes. It is well established that amino acids are compartmentalized using pH gradients generated between organelles and the cytoplasm; however, the dynamics of this partitioning has not been explored. Here we develop a highly sensitive pH reporter and find that the major amino acid storage c...

Significance Genes encoding ribosomal RNA (rDNA) are organized into a repetitive array in eukaryotic genomes. The copy number of these genes often varies and is responsive to genetics and environment. Here, we show that variation in copy number at the rDNA locus is capable of altering replicative lifespan in yeast. These results indicate that consi...

The budding yeast, Saccharomyces cerevisiae , has emerged as a model system for studying the aging processes in eukaryotic cells. However, the full complement of tools available in this organism has not been fully applied, in part because of limitations in throughput that restrict the ability to carry out detailed analyses. Recent advances in micro...

Age-dependent changes in metabolism can manifest as cellular lipid accumulation, but how this accumulation is regulated or impacts longevity is poorly understood. We find that Saccharomyces cerevisiae accumulate lipid droplets (LDs) during aging. We also find that over-expressing BNA2, the first Biosynthesis of NAD⁺ (kynurenine) pathway gene, reduc...

Mitochondrial decline is a hallmark of aging, and cells are equipped with many systems to regulate mitochondrial structure and function in response to stress and metabolic alterations. Here, using budding yeast, we identify a proteolytic pathway that contributes to alterations in mitochondrial structure in aged cells through control of the mitochon...

Interconnectivity and feedback control are hallmarks of biological systems. This includes communication between organelles, which allows them to function and adapt to changing cellular environments. While the specific mechanisms for all communications remain opaque, unraveling the wiring of organelle networks is critical to understand how biologica...

Oligos used in this study. DOI: http://dx.doi.org/10.7554/eLife.13943.021

Complete results of MDC screen. DOI: http://dx.doi.org/10.7554/eLife.13943.019

Yeast strains used in this study. DOI: http://dx.doi.org/10.7554/eLife.13943.020

Significance Long-lived proteins in extracellular spaces (joints/tissues) or within specialized nondividing cells (eye-lens) are associated with age-related decline. However, aging also occurs in dividing stem cells. Although several hypotheses have been proposed to explain how stem cells age, none have addressed whether long-lived proteins contrib...

(A) Yeast Strains. (B) Oligonucleotides. DOI: http://dx.doi.org/10.7554/eLife.03504.010

Mitochondria have a central role in ageing. They are considered to be both a target of the ageing process and a contributor to it. Alterations in mitochondrial structure and function are evident during ageing in most eukaryotes, but how this occurs is poorly understood. Here we identify a functional link between the lysosome-like vacuole and mitoch...

A cytoplasmic protein delivers iron-sulfur clusters to nuclear proteins that function in DNA replication and repair.

Like many asymmetrically dividing cells, budding yeast segregates mitotic spindle poles nonrandomly between mother and daughter cells. During metaphase, the spindle positioning protein Kar9 accumulates asymmetrically, localizing specifically to astral microtubules emanating from the old spindle pole body (SPB) and driving its segregation to the bud...

In realistic ecological and evolutionary systems natural selection acts on multiple levels, i.e. it acts on individuals as well as on collection of individuals. An understanding of evolutionary dynamics of such systems is limited in large part due to the lack of experimental systems that can challenge theoretical models. Mitochondrial genomes (mtDN...

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a syntheti...

Loss of respiratory function is less frequent in MEP strains. A) Examples of inheritance of respiratory function during pedigree analysis of UCC5185. 68% of mothers generate pedigrees with all daughters maintaining respiratory function (upper panel). 32% of mothers lose the ability to produce daughters with respiratory function (small colonies; low...

Preparation and normalization of ERC southern blotting. ERC Southern blot of total genomic DNA isolated from wild type cells and digested with BamHI and with or without RecBCD. Arrow indicates the linear rDNA array. (EPS)

Plasmids used in this study. (DOC)

Yeast strains used in this study. (DOC)

Oligonucleotides used in this study. (DOC)

Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently...

Protein quality control (PQC) degradation systems protect the cell from the toxic accumulation of misfolded proteins. Because any protein can become misfolded, these systems must be able to distinguish abnormal proteins from normal ones, yet be capable of recognizing the wide variety of distinctly shaped misfolded proteins they are likely to encoun...

The dynamic behavior of proteins is critical for cellular homeostasis. However, analyzing dynamics of proteins and protein complexes in vivo has been difficult. Here we describe recombination-induced tag exchange (RITE), a genetic method that induces a permanent epitope-tag switch in the coding sequence after a hormone-induced activation of Cre rec...

The replicative life span (RLS) of Saccharomyces cerevisiae has been established as a model for the genetic regulation of longevity despite the inherent difficulty of the RLS assay, which requires separation of mother and daughter cells by micromanipulation after every division. Here we present the mother enrichment program (MEP), an inducible gene...

The mitochondrial genome (mtDNA) is required for normal cellular function; inherited and somatic mutations in mtDNA lead to a variety of diseases. Saccharomyces cerevisiae has served as a model to study mtDNA integrity, in part because it can survive without mtDNA. A measure of defective mtDNA in S. cerevisiae is the formation of petite colonies. T...

Mutations and deletions in the mitochondrial genome (mtDNA), as well as instability of the nuclear genome, are involved in multiple human diseases. Here, we report that in Saccharomyces cerevisiae, loss of mtDNA leads to nuclear genome instability, through a process of cell-cycle arrest and selection we define as a cellular crisis. This crisis is n...

Individual cells of the budding yeast, Saccharomyces cerevisiae, have a limited life span and undergo a form of senescence termed replicative aging. Replicative life span is defined as the number of daughter cells produced by a yeast mother cell before she ceases dividing. Replicative aging is asymmetric: a mother cell ages but the age of her daugh...

Loss of heterozygosity (LOH) can be a driving force in the evolution of mitotic/somatic diploid cells, and cellular changes that increase the rate of LOH have been proposed to facilitate this process. In the yeast Saccharomyces cerevisiae, spontaneous LOH occurs by a number of mechanisms including chromosome loss and reciprocal and nonreciprocal re...

Dot1 methylates histone H3 lysine 79 (H3K79) on the nucleosome core and is involved in Sir protein-mediated silencing. Previous studies suggested that H3K79 methylation within euchromatin prevents nonspecific binding of the Sir proteins, which in turn facilitates binding of the Sir proteins in unmethylated silent chromatin. However, the mechanism b...

The striking correlation between advanced age and an increased incidence of cancer has led investigators to examine the influence of aging on genome maintenance. Because loss of heterozygosity (LOH) can lead to the inactivation of tumor suppressor genes, and thus carcinogenesis, understanding the affect of aging on this type of mutation event is pa...

Genetic changes increase with the age of organisms, but the basis for this increase is unclear. A study has found that the major pathway of DNA repair is altered with age in the testes of male Drosophila, thus providing a powerful system to dissect the basis for age-related genomic changes.

Yeast pedigree analysis requires a biologist to manually manipulate single yeast cells every 90 minutes for as long as 150 hours. Automating this analysis allows the rapid completion of experiments that would have previously taken a biologist's lifetime. In this paper, progress toward automating yeast pedigree analysis is presented. Also presented...

We previously discovered that the ubiquitin protease Ubp10/Dot4p is important for telomeric silencing through its interaction with Sir4p. However, the mechanism of Ubp10p action was unknown. We now provide evidence that Ubp10p removes ubiquitin from histone H2B; cells with UBP10 deleted have increased steady-state levels of H2B ubiquitination. As a...

Protein quality control degradation systems rid the cell of aberrant proteins, preventing detrimental effects on normal cellular function. Although such systems have been identified in most subcellular compartments, none have been found in the nucleus. Here, we report the discovery of such a system in Saccharomyces cerevisiae. It is defined by San1...

Transcriptional silencing in Saccharomyces requires specific nucleosome modifications promoted in part by a complex of Sir proteins that binds to the modified nucleosomes. Recent evidence suggests that modifications of both the histone amino termini and the core domain of nucleosomes contribute to silencing. We previously identified histone H4 muta...

There is a striking link between increasing age and the incidence of cancer in humans. One of the hallmarks of cancer, genomic instability, has been observed in all types of organisms. In the yeast Saccharomyces cerevisiae, it was recently discovered that during the replicative lifespan, aging cells switch to a state of high genomic instability tha...

The covalent modification of nucleosomal histones has emerged as a major determinant of chromatin structure and gene activity. To understand the interplay between various histone modifications, including acetylation and methylation, we performed a genome-wide chromatin structure analysis in a higher eukaryote. We found a binary pattern of histone m...

Excerpt We are all aware of changes that occur as a personreaches middle age and beyond—wrinkled skin, grayhair, poor vision, etc. But one particularly intriguing phenomenon is the dramatic rise in incidence of cancer withincreasing age: About 75% of all cancers are diagnosedafter the age of 55 (ACS 2004). Because cancer is typically considered a g...

Excerpt When Bruce Stillman made his opening remarks at the69th Cold Spring Harbor Symposium, one of the thingshe said he hoped to learn was a way to easily explain what"epigenetics" meant to his wife Grace. After a week ofdiscussions, it became clear that such a request was akinto asking someone to define "family values"—everyoneknew what it meant...

Yeast pedigree analysis-isolation and characterization of the products of mitotic cell divisions throughout the lifespan of an individual cell-is a manually intensive process that requires a biologist to manipulate single yeast cells every 90 minutes for as long as 150 hours. Progress toward the development of a system for automating yeast pedigree...

Ku is a conserved DNA end-binding protein that plays various roles at different kinds of DNA ends. At telomeres, Ku is part of the structure that protects the chromosome end, whereas at broken DNA ends, Ku promotes DNA repair as part of the nonhomologous end-joining (NHEJ) pathway. Here, we present evidence of a new role for Ku that impacts both te...

There is a strong correlation between age and cancer, but the mechanism by which this phenomenon occurs is unclear. We chose Saccharomyces cerevisiae to examine one of the hallmarks of cancer--genomic instability--as a function of cellular age. As diploid yeast mother cells aged, an approximately 100-fold increase in loss of heterozygosity (LOH) oc...

Electrospray ionization mass spectrometry, a leading method for the quantification of biomolecules, is useful for the analysis of posttranslational modifications of proteins. Here we describe a mass spectrometric approach for determining levels of acetylation at individual lysine residues within the amino-terminal tail of histone H4. Because of the...

In this issue of Cell, Meneghini et al. demonstrate that replacement of canonical histone H2A with the histone variant H2A.Z within euchromatin prevents silent chromatin proteins from migrating into regions of the chromosome that normally are transcriptionally active. Thus, this highly conserved histone variant is critical for defining chromatin do...

In this issue of Cell, Meneghini et al. demonstrate that replacement of canonical histone H2A with the histone variant H2A.Z within euchromatin prevents silent chromatin proteins from migrating into regions of the chromosome that normally are transcriptionally active. Thus, this highly conserved histone variant is critical for defining chromatin do...

More than 20 residues within the four core histone proteins of the nucleosome are potential sites of post-translational modifications, such as methylation, acetylation, ubiquitination and phosphorylation. It has been hypothesized that specific patterns of these modifications on the nucleosome facilitate recruitment of non-histone proteins to chroma...

Telomeric position effect in Saccharomyces cerevisiae is a chromatin-mediated phenomenon in which telomere proximal genes are repressed (silenced) in a heritable, but reversible, fashion. Once a transcriptional state (active or silenced) is established, however, there is a strong tendency for that state to be propagated. Twenty-five years ago, H. W...

Telomeres are the protective ends of linear chromosomes. Telomeric components have been identified and described by their abilities to bind telomeric DNA, affect telomere repeat length, participate in telomeric DNA replication, or modulate transcriptional silencing of telomere-adjacent genes; however, their roles in chromosome end protection are no...

DOT1 was originally identified as a gene affecting telomeric silencing in S. cerevisiae. We now find that Dot1p methylates histone H3 on lysine 79, which maps to the top and bottom of the nucleosome core. Methylation occurs only when histone H3 is assembled in chromatin. In vivo, Dot1p is solely responsible for this methylation and methylates appro...

Silencing in Saccharomyces cerevisiae is a chromatin-mediated repression of genes located within specific chromosomal domains. Each time chromosomal DNA replicates, silent chromatin must be recreated by an orchestrated series of assembly processes and events. Mutations affecting DNA replication, cell cycle progression, DNA repair, chromatin assembl...

Sir2p is an NAD(+)-dependent histone deacetylase required for chromatin-dependent silencing in yeast. In a cell-based screen for inhibitors of Sir2p, we identified a compound, splitomicin, that creates a conditional phenocopy of a sir2 deletion mutant in Saccharomyces cerevisiae. Cells grown in the presence of the drug have silencing defects at tel...

The Saccharomyces cerevisiae Mre11p/Rad50p/Xrs2p (MRX) complex is evolutionarily conserved and functions in DNA repair and at telomeres [1-3]. In vivo, MRX is required for a 5' --> 3' exonuclease activity that mediates DNA recombination at double-strand breaks (DSBs). Paradoxically, abolition of this exonuclease activity in MRX mutants results in s...

The telomerase enzyme lengthens telomeres, an activity essential for chromosome stability in most eukaryotes. The enzyme is composed of a specialized reverse transcriptase and a template RNA. In Saccharomyces cerevisiae, overexpression of TLC1, the telomerase RNA gene, disrupts telomeric structure. The result is both shortened telomere length and l...

There are still many mysteries surrounding how silenced regions of the eukaryotic genome are created and maintained. But recent discoveries about the most evolutionarily conserved silencing protein, Sir2p, have provided new mechanistic insights into these processes.

Hat1p and Hat2p are the two subunits of a type B histone acetyltransferase from Saccharomyces cerevisiae that acetylates free histone H4 on lysine 12 in vitro. However, the role for these gene products in chromatin function has been unclear, as deletions of the HAT1 and/or HAT2 gene displayed no obvious phenotype. We have now identified a role for...

Excerpt Pioneering experiments by H.J. Muller (1938) and Barbara McClintock (1941) defined telomeres as the "caps"of linear eukaryotic chromosomes. Whereas broken DNAends are susceptible to degradation and end-to-end fusions, telomeres are resistant to these activities. Overtime, the field of telomere biology has tried to understandwhat makes nativ...

To better understand the requirements for telomerase-mediated telomere addition in vivo, we developed an assay in S. cerevisiae that creates a chromosome end immediately adjacent to a short telomeric DNA tract. The de novo end acts as a telomere: it is protected from degradation in a CDC13-dependent manner, telomeric sequences are added efficiently...

Transcriptional silencing in Saccharomyces cerevisiaeoccurs at specific loci and is mediated by a multiprotein complex that includes Rap1p and the Sir proteins. We studied the function of a recently identified gene, DOT4, that disrupts silencing when overexpressed. DOT4 encodes an ubiquitin processing protease (hydrolase) that is primarily located...

Telomeres impart stability on linear eukaryotic chromosomes by acting as caps, preventing chromosomes from fusing together or being degraded. The structure of a telomere end binding protein in a complex with DNA provides the first molecular view of chromosome capping.

Saccharomyces cerevisiae telomeric DNA replicates late in S phase, and telomeric genes are transcriptionally silent. Transcriptional repression of telomere-proximal genes results from silent chromatin initiating at the chromosome end, but the relationship between telomeric chromatin and DNA replication is unknown. Mutations in SIR3, a silent chroma...

The ends of chromosomes in Saccharomyces cerevisiae initiate a repressive chromatin structure that spreads internally and inhibits the transcription of nearby genes, a phenomenon termed telomeric silencing. To investigate the molecular basis of this process, we carried out a genetic screen to identify genes whose overexpression disrupts telomeric s...

Sister chromatid cohesion and chromosome condensation are both essential to the successful completion of mitosis. The recent identification and characterization of the yeast Mcd1p/Scc1p protein reveals a previously unsuspected mechanistic link between these processes.

It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres. This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci. Here, we show that a null mutation of the DNA repair gene RAD6...

We have isolated the predominant cytoplasmic histone acetyltransferase activity from Saccharomyces cerevisiae. This enzyme acetylates the lysine at residue 12 of free histone H4 but does not modify histone H4 when packaged in chromatin. The activity contains two proteins, Hat1p and Hat2p. Hat1p is the catalytic subunit of the histone acetyltransfer...

Telomeres are required for the stable maintenance of chromosomes in the yeast Saccharomyces cerevisiae. Telomeres also repress the expression of genes in their vicinity, a phenomenon known as telomere position effect. In an attempt to construct a conditional telomere, an inducible promoter was introduced adjacent to a single telomere of a chromosom...

Telomeres, the natural ends of linear eukaryotic chromosomes, are essential for chromosome stability. Because of the nature of DNA replication, telomeres require a specialized mechanism to ensure their complete duplication. Telomeres are also capable of silencing the transcription of genes that are located near them. In order to identify genes in t...

Genes located near telomeres in yeast are subject to position-effect variegation. To better understand the mechanism of this variegation, we investigated how a telomeric URA3 gene switches from a silent to an expressed state. We found that silencing of a telomeric URA3 gene was attributable to the elimination of its basal transcription. The reversa...

In Saccharomyces cerevisiae, telomeres repress transcription of genes located nearby. This region-specific gene inactivation is thought to involve the packaging of telomeric domains into silent chromatin. To gain insight into the mechanism of telomeric silencing, a genetic assay to examine the spread of silencing along the distal right arm of chrom...

Genes located near telomeres in Saccharomyces cerevisiae undergo position-effect variegation; their transcription is subject to reversible but mitotically heritable repression. This position effect and the finding that telomeric DNA is late replicating suggest that yeast telomeres exist in a heterochromatin-like state. Mutations in genes that suppr...

The chromatin structures of the telomeric and subtelomeric regions on chromosomal DNA molecules in Saccharomyces cerevisiae were analyzed using micrococcal nuclease and DNAse I. The subtelomeric repeats X and Y' were assembled in nucleosomes. However, the terminal tracts of C1-3A repeats were protein protected in a particle larger than a nucleosome...

Genes placed near telomeres in S. cerevisiae succumb to position-effect variegation. SIR2, SIR3, SIR4, NAT1, ARD1, and HHF2 (histone H4) were identified as modifiers of the position effect at telomeres, since transcriptional repression near telomeres was no longer observed when any of the modifier genes were mutated. These genes, in addition to SIR...

S. cerevisiae chromosomes end with the telomeric repeat (TG1-3)n. When any of four Pol II genes was placed immediately adjacent to the telomeric repeats, expression of the gene was reversibly repressed as demonstrated by phenotype and mRNA analyses. For example, cells bearing a telomere-linked copy of ADE2 produced predominantly red colonies (a phe...

This chapter discusses the DNA–protein complexes found at telomeres in the macronuclei of ciliated protozoans, with special attention given to studies in Oxytricha (a hypotrichous ciliate) and Tetrahymena (a holotrichous ciliate). The structure of telomeres has been largely elucidated by studies on macronuclear DNA from ciliated protozoans. Studies...

The macronuclear DNA in the ciliated protozoan O. nova consists of integral of 10(7) gene-sized DNA molecules, all of which terminate with 20 bp of C4A4 repeats followed by a 3' (G4T4)2 single-stranded tail. Two immunologically distinct proteins of 55 and 26 kd, which are tenaciously, but noncovalently associated with Oxytricha macronuclear DNA ter...

Oxytricha macronuclear DNA exists as approximately 24 X 10(6) gene-sized molecules terminating with a C4A4 repeat. DNA-protein interactions at the ends of bulk macronuclear molecules were probed with micrococcal nuclease and methidiumpropyl-EDTA X Fe(II) (MPE X Fe[II]). The ends were indirectly labeled by hybridizing with (C4A4)2. Alternatively, a...

The chromatin structure of the palindromic macronuclear ribosomal RNA genes of Tetrahymena thermophila was probed with micrococcal nuclease. Independent of the state of transcriptional activity, the transcribed region had a shorter nucleosome repeat (184±3 base pairs) than the non-transcribed central spacer or bulk chromatin (both 200 base pairs)....

In the macronuclear rRNA genes of Tetrahymena thermophila, a 413 bp intervening sequence (IVS) interrupts the 26S rRNA-coding region. A restriction fragment of the rDNA containing the IVS and portions of the adjacent rRNA sequences (exons) was inserted downstream from the lac UV5 promoter in a recombinant plasmid. Transcription of this template by...

Thesis (M.S.)--University of Colorado at Boulder, 1980. Includes bibliographical references (leaves 45-47).