Itay Tirosh | Broad Institute (original) (raw)

Papers by Itay Tirosh

Genome research, 2015

Both intrinsic cell state changes and variations in the composition of stem cell populations have... more Both intrinsic cell state changes and variations in the composition of stem cell populations have been implicated as contributors to aging. We used single cell RNA-seq to dissect variability in hematopoietic stem and progenitor cell populations from young and old mice from two strains. We found that cell cycle dominates the variability within each population, and that there is a lower frequency of cells in the G1 phase among old compared to young long-term hematopoietic stem cells, suggesting that they traverse through G1 faster. Moreover, transcriptional changes in HSCs during aging are inversely related to those upon HSC differentiation, such that old short term (ST)-HSCs resemble young long-term (LT-HSCs), suggesting that they exist in a less differentiated state. Our results indicate both compositional changes and intrinsic, population-wide changes with age and are consistent with a model where a relationship between cell-cycle progression and self-renewal versus differentiation...

Cell, 2015

Finding the components of cellular circuits and determining their functions systematically remain... more Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits.

Cell, 2015

Cells, the basic units of biological structure and function, vary broadly in type and state. Sing... more Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. VIDEO ABSTRACT.

G3 (Bethesda, Md.), Jan 5, 2015

Previous studies have described a transcriptional "memory effect", whereby transcript l... more Previous studies have described a transcriptional "memory effect", whereby transcript levels of many Abf1-regulated genes in the budding yeast Saccharomyces cerevisiae are undiminished even after Abf1 has dissociated from its regulatory sites. Here we provide additional support for this effect and investigate its molecular basis. We show that the effect is observed in a distinct abf1 ts mutant from that used in earlier studies, demonstrating that it is robust, and use chromatin immunoprecipitation (ChIP) to show that Abf1 association is decreased similarly from memory effect and transcriptionally responsive genes at the restrictive temperature. We also demonstrate that the association of TBP and Pol II decreases following loss of Abf1 binding for transcriptionally responsive genes but not for memory effect genes. Examination of genome-wide nucleosome occupancy data reveals that while transcriptionally responsive genes exhibit increased nucleosome occupancy in abf1 ts yeast...

BMC bioinformatics, 2005

High-throughput methods identify an overwhelming number of protein-protein interactions. However,... more High-throughput methods identify an overwhelming number of protein-protein interactions. However, the limited accuracy of these methods results in the false identification of many spurious interactions. Accordingly, the resulting interactions are regarded as hypothetical and computational methods are needed to increase their confidence. Several methods have recently been suggested for this purpose including co-expression as a confidence measure for interacting proteins, but their performance is still quite poor. We introduce a novel computational method for verification of protein-protein interactions based on the co-expression of orthologs of interacting partners. The performance of our method is analysed using known S. cerevisiae interactions, and is shown to overcome limitations of previous methods. We present specific examples of known and putative interactions that are detected by our method and not by previous methods, and suggest that they represent transient interactions tha...

PLoS Genetics, 2012

Understanding why genes evolve at different rates is fundamental to evolutionary thinking. In spe... more Understanding why genes evolve at different rates is fundamental to evolutionary thinking. In species of the budding yeast, the rate at which genes diverge in expression correlates with the organization of their promoter nucleosomes: genes lacking a nucleosome-free region (denoted OPN for ''Occupied Proximal Nucleosomes'') vary widely between the species, while the expression of those containing NFR (denoted DPN for ''Depleted Proximal Nucleosomes'') remains largely conserved. To examine if early evolutionary dynamics contributes to this difference in divergence, we artificially selected for high expression of GFP-fused proteins. Surprisingly, selection was equally successful for OPN and DPN genes, with ,80% of genes in each group stably increasing in expression by a similar amount. Notably, the two groups adapted by distinct mechanisms: DPN-selected strains duplicated large genomic regions, while OPN-selected strains favored trans mutations not involving duplications. When selection was removed, DPN (but not OPN) genes reverted rapidly to wild-type expression levels, consistent with their lower diversity between species. Our results suggest that promoter organization constrains the early evolutionary dynamics and in this way biases the path of long-term evolution.

Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the... more Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres) was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC) components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.

Genome Biology, 2010

In yeast, the chromatin remodeler Isw1 shifts nucleosomes from mid-coding, to more 5' regions of ... more In yeast, the chromatin remodeler Isw1 shifts nucleosomes from mid-coding, to more 5' regions of genes and may regulate transcrip-tional elongation.

Journal of Biology, 2009

Evolutionary changes in gene expression are a main driver of phenotypic evolution. In yeast, gene... more Evolutionary changes in gene expression are a main driver of phenotypic evolution. In yeast, genes that have rapidly diverged in expression are associated with particular promoter features, including the presence of a TATA box, a nucleosome-covered promoter and unstable tracts of tandem repeats. Here, we discuss how these promoter properties may confer an inherent capacity for flexibility of expression.

Genome Biology, 2007

Background: Gene duplication provides raw material for the generation of new functions, but most ... more Background: Gene duplication provides raw material for the generation of new functions, but most duplicates are rapidly lost due to the initial redundancy in gene function. How gene function diversifies following duplication is largely unclear. Previous studies analyzed the diversification of duplicates by characterizing their coding sequence divergence. However, functional divergence can also be attributed to changes in regulatory properties, such as protein localization or expression, which require only minor changes in gene sequence.

Trends in Genetics, 2008

Absence of daily cycles in plasma sex steroids in male and female tuatara (Sphenodon punctatus), ... more Absence of daily cycles in plasma sex steroids in male and female tuatara (Sphenodon punctatus), and the effects of acute capture stress on females. Gen. Comp. Endocrinol. 79, 103-113 4 Cree, A. et al. (1991) Laparoscopy, radiography, and blood analyses as techniques for identifying the reproductive condition of female tuatara. The rate of DNA evolution: effects of body size and temperature on the molecular clock. Body size, metabolicrate, generation time, and the molecular clock. Proc. Natl. Acad. Sci. U. S. A. 90, 4087-4091 8 Quinn, T.W. (1992) The genetic legacy of mother goose: phylogeographic patterns of lesser snow goose Chen caerulescens caerulescens maternal lineages. Mol. Ecol. 1, 105-117 9 Stoneking, M. et al. (1992) New approaches to dating suggest a recent age for the human mtDNA ancestor.

Science, 2009

The following resources related to this article are available online at

Proceedings of the National Academy of Sciences, 2011

Closely related species show a high degree of differences in gene expression, but the functional ... more Closely related species show a high degree of differences in gene expression, but the functional significance of these differences remains unclear. Similarly, stress responses in yeast typically involve differential expression of numerous genes, and it is unclear how many of these are functionally significant. To address these issues, we compared the expression programs of four yeast species under different growth conditions, and found that the response of these species to stress has diverged extensively. On an individual gene basis, most transcriptional responses are not conserved in any pair of species, and there are very limited common responses among all four species. We present evidence that many evolutionary changes in stress responses are compensated either (i) by the response of related genes or (ii) by changes in the basal expression levels of the genes whose responses have diverged. Thus, stress-related genes are often induced upon stress in some species but maintain high levels even in the absence of stress at other species, indicating a transition between induced and constitutive activation. In addition, ∼15% of the stress responses are specific to only one of the four species, with no evidence for compensating effects or stress-related annotations, and these may reflect fortuitous regulation that is unimportant for the stress response (i.e., biological noise). Frequent compensatory changes and biological noise may explain how diverged expression responses support similar physiological responses. evolutionary conservation | functional evolution | gene regulation |

PLoS Biology, 2011

mRNA levels are determined by the balance between transcription and mRNA degradation, and while t... more mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast.

Molecular Systems Biology, 2010

Gene regulation differs greatly between related species, constituting a major source of phenotypi... more Gene regulation differs greatly between related species, constituting a major source of phenotypic diversity. Recent studies characterized extensive differences in the gene expression programs of closely related species. In contrast, virtually nothing is known about the evolution of chromatin structure and how it influences the divergence of gene expression. Here, we compare the genomewide nucleosome positioning of two closely related yeast species and, by profiling their interspecific hybrid, trace the genetic basis of the observed differences into mutations affecting the local DNA sequences (cis effects) or the upstream regulators (trans effects). The majority (B70%) of inter-species differences is due to cis effects, leaving a significant contribution (30%) for trans factors. We show that cis effects are well explained by mutations in nucleosome-disfavoring AT-rich sequences, but are not associated with divergence of nucleosome-favoring sequences. Differences in nucleosome positioning propagate to multiple adjacent nucleosomes, supporting the statistical positioning hypothesis, and we provide evidence that nucleosome-free regions, but not the þ 1 nucleosome, serve as stable border elements. Surprisingly, although we find that differential nucleosome positioning among cell types is strongly correlated with differential expression, this does not seem to be the case for evolutionary changes: divergence of nucleosome positioning is excluded from regulatory elements and is not correlated with gene expression divergence, suggesting a primarily neutral mode of evolution. Our results provide evolutionary insights to the genetic determinants and regulatory function of nucleosome positioning.

Molecular Systems Biology, 2008

Recent studies have characterized significant differences in the cis-regulatory sequences of rela... more Recent studies have characterized significant differences in the cis-regulatory sequences of related organisms, but the impact of these differences on gene expression remains largely unexplored. Here, we show that most previously identified differences in transcription factor (TF)-binding sequences of yeasts and mammals have no detectable effect on gene expression, suggesting that compensatory mechanisms allow promoters to rapidly evolve while maintaining a stabilized expression pattern. To examine the impact of changes in cis-regulatory elements in a more controlled setting, we compared the genes induced during mating of three yeast species. This response is governed by a single TF (STE12), and variations in its predicted binding sites can indeed account for about half of the observed expression differences. The remaining unexplained differences are correlated with the increased divergence of the sequences that flank the binding sites and an apparent modulation of chromatin structure. Our analysis emphasizes the flexibility of promoter structure, and highlights the interplay between specific binding sites and general chromatin structure in the control of gene expression.

Genome research, 2015

Both intrinsic cell state changes and variations in the composition of stem cell populations have... more Both intrinsic cell state changes and variations in the composition of stem cell populations have been implicated as contributors to aging. We used single cell RNA-seq to dissect variability in hematopoietic stem and progenitor cell populations from young and old mice from two strains. We found that cell cycle dominates the variability within each population, and that there is a lower frequency of cells in the G1 phase among old compared to young long-term hematopoietic stem cells, suggesting that they traverse through G1 faster. Moreover, transcriptional changes in HSCs during aging are inversely related to those upon HSC differentiation, such that old short term (ST)-HSCs resemble young long-term (LT-HSCs), suggesting that they exist in a less differentiated state. Our results indicate both compositional changes and intrinsic, population-wide changes with age and are consistent with a model where a relationship between cell-cycle progression and self-renewal versus differentiation...

Cell, 2015

Finding the components of cellular circuits and determining their functions systematically remain... more Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits.

Cell, 2015

Cells, the basic units of biological structure and function, vary broadly in type and state. Sing... more Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. VIDEO ABSTRACT.

G3 (Bethesda, Md.), Jan 5, 2015

Previous studies have described a transcriptional "memory effect", whereby transcript l... more Previous studies have described a transcriptional "memory effect", whereby transcript levels of many Abf1-regulated genes in the budding yeast Saccharomyces cerevisiae are undiminished even after Abf1 has dissociated from its regulatory sites. Here we provide additional support for this effect and investigate its molecular basis. We show that the effect is observed in a distinct abf1 ts mutant from that used in earlier studies, demonstrating that it is robust, and use chromatin immunoprecipitation (ChIP) to show that Abf1 association is decreased similarly from memory effect and transcriptionally responsive genes at the restrictive temperature. We also demonstrate that the association of TBP and Pol II decreases following loss of Abf1 binding for transcriptionally responsive genes but not for memory effect genes. Examination of genome-wide nucleosome occupancy data reveals that while transcriptionally responsive genes exhibit increased nucleosome occupancy in abf1 ts yeast...

BMC bioinformatics, 2005

High-throughput methods identify an overwhelming number of protein-protein interactions. However,... more High-throughput methods identify an overwhelming number of protein-protein interactions. However, the limited accuracy of these methods results in the false identification of many spurious interactions. Accordingly, the resulting interactions are regarded as hypothetical and computational methods are needed to increase their confidence. Several methods have recently been suggested for this purpose including co-expression as a confidence measure for interacting proteins, but their performance is still quite poor. We introduce a novel computational method for verification of protein-protein interactions based on the co-expression of orthologs of interacting partners. The performance of our method is analysed using known S. cerevisiae interactions, and is shown to overcome limitations of previous methods. We present specific examples of known and putative interactions that are detected by our method and not by previous methods, and suggest that they represent transient interactions tha...

PLoS Genetics, 2012

Understanding why genes evolve at different rates is fundamental to evolutionary thinking. In spe... more Understanding why genes evolve at different rates is fundamental to evolutionary thinking. In species of the budding yeast, the rate at which genes diverge in expression correlates with the organization of their promoter nucleosomes: genes lacking a nucleosome-free region (denoted OPN for ''Occupied Proximal Nucleosomes'') vary widely between the species, while the expression of those containing NFR (denoted DPN for ''Depleted Proximal Nucleosomes'') remains largely conserved. To examine if early evolutionary dynamics contributes to this difference in divergence, we artificially selected for high expression of GFP-fused proteins. Surprisingly, selection was equally successful for OPN and DPN genes, with ,80% of genes in each group stably increasing in expression by a similar amount. Notably, the two groups adapted by distinct mechanisms: DPN-selected strains duplicated large genomic regions, while OPN-selected strains favored trans mutations not involving duplications. When selection was removed, DPN (but not OPN) genes reverted rapidly to wild-type expression levels, consistent with their lower diversity between species. Our results suggest that promoter organization constrains the early evolutionary dynamics and in this way biases the path of long-term evolution.

Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the... more Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres) was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC) components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.

Genome Biology, 2010

In yeast, the chromatin remodeler Isw1 shifts nucleosomes from mid-coding, to more 5' regions of ... more In yeast, the chromatin remodeler Isw1 shifts nucleosomes from mid-coding, to more 5' regions of genes and may regulate transcrip-tional elongation.

Journal of Biology, 2009

Evolutionary changes in gene expression are a main driver of phenotypic evolution. In yeast, gene... more Evolutionary changes in gene expression are a main driver of phenotypic evolution. In yeast, genes that have rapidly diverged in expression are associated with particular promoter features, including the presence of a TATA box, a nucleosome-covered promoter and unstable tracts of tandem repeats. Here, we discuss how these promoter properties may confer an inherent capacity for flexibility of expression.

Genome Biology, 2007

Background: Gene duplication provides raw material for the generation of new functions, but most ... more Background: Gene duplication provides raw material for the generation of new functions, but most duplicates are rapidly lost due to the initial redundancy in gene function. How gene function diversifies following duplication is largely unclear. Previous studies analyzed the diversification of duplicates by characterizing their coding sequence divergence. However, functional divergence can also be attributed to changes in regulatory properties, such as protein localization or expression, which require only minor changes in gene sequence.

Trends in Genetics, 2008

Absence of daily cycles in plasma sex steroids in male and female tuatara (Sphenodon punctatus), ... more Absence of daily cycles in plasma sex steroids in male and female tuatara (Sphenodon punctatus), and the effects of acute capture stress on females. Gen. Comp. Endocrinol. 79, 103-113 4 Cree, A. et al. (1991) Laparoscopy, radiography, and blood analyses as techniques for identifying the reproductive condition of female tuatara. The rate of DNA evolution: effects of body size and temperature on the molecular clock. Body size, metabolicrate, generation time, and the molecular clock. Proc. Natl. Acad. Sci. U. S. A. 90, 4087-4091 8 Quinn, T.W. (1992) The genetic legacy of mother goose: phylogeographic patterns of lesser snow goose Chen caerulescens caerulescens maternal lineages. Mol. Ecol. 1, 105-117 9 Stoneking, M. et al. (1992) New approaches to dating suggest a recent age for the human mtDNA ancestor.

Science, 2009

The following resources related to this article are available online at

Proceedings of the National Academy of Sciences, 2011

Closely related species show a high degree of differences in gene expression, but the functional ... more Closely related species show a high degree of differences in gene expression, but the functional significance of these differences remains unclear. Similarly, stress responses in yeast typically involve differential expression of numerous genes, and it is unclear how many of these are functionally significant. To address these issues, we compared the expression programs of four yeast species under different growth conditions, and found that the response of these species to stress has diverged extensively. On an individual gene basis, most transcriptional responses are not conserved in any pair of species, and there are very limited common responses among all four species. We present evidence that many evolutionary changes in stress responses are compensated either (i) by the response of related genes or (ii) by changes in the basal expression levels of the genes whose responses have diverged. Thus, stress-related genes are often induced upon stress in some species but maintain high levels even in the absence of stress at other species, indicating a transition between induced and constitutive activation. In addition, ∼15% of the stress responses are specific to only one of the four species, with no evidence for compensating effects or stress-related annotations, and these may reflect fortuitous regulation that is unimportant for the stress response (i.e., biological noise). Frequent compensatory changes and biological noise may explain how diverged expression responses support similar physiological responses. evolutionary conservation | functional evolution | gene regulation |

PLoS Biology, 2011

mRNA levels are determined by the balance between transcription and mRNA degradation, and while t... more mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast.

Molecular Systems Biology, 2010

Gene regulation differs greatly between related species, constituting a major source of phenotypi... more Gene regulation differs greatly between related species, constituting a major source of phenotypic diversity. Recent studies characterized extensive differences in the gene expression programs of closely related species. In contrast, virtually nothing is known about the evolution of chromatin structure and how it influences the divergence of gene expression. Here, we compare the genomewide nucleosome positioning of two closely related yeast species and, by profiling their interspecific hybrid, trace the genetic basis of the observed differences into mutations affecting the local DNA sequences (cis effects) or the upstream regulators (trans effects). The majority (B70%) of inter-species differences is due to cis effects, leaving a significant contribution (30%) for trans factors. We show that cis effects are well explained by mutations in nucleosome-disfavoring AT-rich sequences, but are not associated with divergence of nucleosome-favoring sequences. Differences in nucleosome positioning propagate to multiple adjacent nucleosomes, supporting the statistical positioning hypothesis, and we provide evidence that nucleosome-free regions, but not the þ 1 nucleosome, serve as stable border elements. Surprisingly, although we find that differential nucleosome positioning among cell types is strongly correlated with differential expression, this does not seem to be the case for evolutionary changes: divergence of nucleosome positioning is excluded from regulatory elements and is not correlated with gene expression divergence, suggesting a primarily neutral mode of evolution. Our results provide evolutionary insights to the genetic determinants and regulatory function of nucleosome positioning.

Molecular Systems Biology, 2008

Recent studies have characterized significant differences in the cis-regulatory sequences of rela... more Recent studies have characterized significant differences in the cis-regulatory sequences of related organisms, but the impact of these differences on gene expression remains largely unexplored. Here, we show that most previously identified differences in transcription factor (TF)-binding sequences of yeasts and mammals have no detectable effect on gene expression, suggesting that compensatory mechanisms allow promoters to rapidly evolve while maintaining a stabilized expression pattern. To examine the impact of changes in cis-regulatory elements in a more controlled setting, we compared the genes induced during mating of three yeast species. This response is governed by a single TF (STE12), and variations in its predicted binding sites can indeed account for about half of the observed expression differences. The remaining unexplained differences are correlated with the increased divergence of the sequences that flank the binding sites and an apparent modulation of chromatin structure. Our analysis emphasizes the flexibility of promoter structure, and highlights the interplay between specific binding sites and general chromatin structure in the control of gene expression.