Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome.

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these "hungry spliceosome" conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under positive selection and are fated to disappear. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of "intronization", whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function

Specificity in Transcriptional Regulation

Gene-specific regulation of transcription is achieved through the binding of transcription factors to DNA sequences. Many Eukaryotic transcription factors maintain affinity differences between target and non-target sequences that appear too small to explain the specificity observed for the genes they regulate. How is specificity achieved in Eukaryotic gene expression? In eukaryotes, DNA is spooled around histone protein octamers to form nucleosomes. The nucleosome represses transcription by acting as a barrier to the binding of transcription factors. Thus, gene activation requires the recruitment of ATP-dependent chromatin remodelers which remove nucleosomes covering important regulatory sequences. However, promoter nucleosome structure is heterogeneous even under activating conditions. Why does the cell expend energy to maintain heterogenous promoter chromatin in the promoters of actively transcribing genes? In Chapter 1, I present a model of gene transcription which represents a unified solution to these questions, among others. I show that activator mediated ATP dependent stochastic removal and reformation of nucleosomes on promoter DNA may be used for the kinetic proofreading of activator-DNA interactions. The specificity enhancement due to kinetic proofreading is an archetype that, in part, can be used to explain the observed specificity in Eukaryotic gene expression. I show that contrary to expectation, heterogeneity in promoter chromatin structure reduces the variation observed in gene expression. Additionally, I provide insight into the necessity of transcriptional bursting for regulated, highly expressed genes.In Chapter 2, I present a number of experimental tests of the proofreading model. We observe transcriptional bursting, chromatin remodeling and activator binding at a classic model gene, PHO5, in Saccharomyces cerevisiae. I show that transcriptional bursting of PHO5 occurs in at least two distinct timescales, an expectation of the proofreading model. In addition, I show that mutation of a single chromatin remodeler, Isw2, is sufficient to disrupt correlation at the longer timescale. I present a model of kinetic proofreading of activator specificity by Isw2 and test conjectures such a model purports.In chapter 3, I present a technique for studying eukaryotic gene expression by generating and testing the expression of >400,000 permuted synthetic cassettes generated from 26 genes from Saccharomyces cerevisiae

Specificity in Transcriptional Regulation

Gene-specific regulation of transcription is achieved through the binding of transcription factors to DNA sequences. Many Eukaryotic transcription factors maintain affinity differences between target and non-target sequences that appear too small to explain the specificity observed for the genes they regulate. How is specificity achieved in Eukaryotic gene expression? In eukaryotes, DNA is spooled around histone protein octamers to form nucleosomes. The nucleosome represses transcription by acting as a barrier to the binding of transcription factors. Thus, gene activation requires the recruitment of ATP-dependent chromatin remodelers which remove nucleosomes covering important regulatory sequences. However, promoter nucleosome structure is heterogeneous even under activating conditions. Why does the cell expend energy to maintain heterogenous promoter chromatin in the promoters of actively transcribing genes? In Chapter 1, I present a model of gene transcription which represents a unified solution to these questions, among others. I show that activator mediated ATP dependent stochastic removal and reformation of nucleosomes on promoter DNA may be used for the kinetic proofreading of activator-DNA interactions. The specificity enhancement due to kinetic proofreading is an archetype that, in part, can be used to explain the observed specificity in Eukaryotic gene expression. I show that contrary to expectation, heterogeneity in promoter chromatin structure reduces the variation observed in gene expression. Additionally, I provide insight into the necessity of transcriptional bursting for regulated, highly expressed genes.In Chapter 2, I present a number of experimental tests of the proofreading model. We observe transcriptional bursting, chromatin remodeling and activator binding at a classic model gene, PHO5, in Saccharomyces cerevisiae. I show that transcriptional bursting of PHO5 occurs in at least two distinct timescales, an expectation of the proofreading model. In addition, I show that mutation of a single chromatin remodeler, Isw2, is sufficient to disrupt correlation at the longer timescale. I present a model of kinetic proofreading of activator specificity by Isw2 and test conjectures such a model purports.In chapter 3, I present a technique for studying eukaryotic gene expression by generating and testing the expression of >400,000 permuted synthetic cassettes generated from 26 genes from Saccharomyces cerevisiae

Nucleosomal proofreading of activator–promoter interactions

Specificity in transcriptional regulation is imparted by transcriptional activators that bind to specific DNA sequences from which they stimulate transcription. Specificity may be increased by slowing down the kinetics of regulation: by increasing the energy for dissociation of the activator-DNA complex or decreasing activator concentration. In general, higher dissociation energies imply longer DNA dwell times of the activator; the activator-bound gene may not readily turn off again. Lower activator concentrations entail longer pauses between binding events; the activator-unbound gene is not easily turned on again and activated transcription occurs in stochastic bursts. We show that kinetic proofreading of activator-DNA recognition-insertion of an energy-dissipating delay step into the activation pathway for transcription-reconciles high specificity of transcriptional regulation with fast regulatory kinetics. We show that kinetic proofreading results from the stochastic removal and reformation of promoter nucleosomes, at a distance from equilibrium

From Structural Variation of Gene Molecules to Chromatin Dynamics and Transcriptional Bursting.

Transcriptional activation of eukaryotic genes is accompanied, in general, by a change in the sensitivity of promoter chromatin to endonucleases. The structural basis of this alteration has remained elusive for decades; but the change has been viewed as a transformation of one structure into another, from "closed" to "open" chromatin. In contradistinction to this static and deterministic view of the problem, a dynamical and probabilistic theory of promoter chromatin has emerged as its solution. This theory, which we review here, explains observed variation in promoter chromatin structure at the level of single gene molecules and provides a molecular basis for random bursting in transcription-the conjecture that promoters stochastically transition between transcriptionally conducive and inconducive states. The mechanism of transcriptional regulation may be understood only in probabilistic terms

From Structural Variation of Gene Molecules to Chromatin Dynamics and Transcriptional Bursting.

Transcriptional activation of eukaryotic genes is accompanied, in general, by a change in the sensitivity of promoter chromatin to endonucleases. The structural basis of this alteration has remained elusive for decades; but the change has been viewed as a transformation of one structure into another, from "closed" to "open" chromatin. In contradistinction to this static and deterministic view of the problem, a dynamical and probabilistic theory of promoter chromatin has emerged as its solution. This theory, which we review here, explains observed variation in promoter chromatin structure at the level of single gene molecules and provides a molecular basis for random bursting in transcription-the conjecture that promoters stochastically transition between transcriptionally conducive and inconducive states. The mechanism of transcriptional regulation may be understood only in probabilistic terms

Permutational analysis of Saccharomyces cerevisiae regulatory elements.

Gene expression in Saccharomyces cerevisiae is regulated at multiple levels. Genomic and epigenomic mapping of transcription factors and chromatin factors has led to the delineation of various modular regulatory elements-enhancers (upstream activating sequences), core promoters, 5' untranslated regions (5' UTRs) and transcription terminators/3' untranslated regions (3' UTRs). However, only a few of these elements have been tested in combinations with other elements and the functional interactions between the different modular regulatory elements remain under explored. We describe a simple and rapid approach to build a combinatorial library of regulatory elements and have used this library to study 26 different enhancers, core promoters, 5' UTRs and transcription terminators/3' UTRs to estimate the contribution of individual regulatory parts in gene expression. Our combinatorial analysis shows that while enhancers initiate gene expression, core promoters modulate the levels of enhancer-mediated expression and can positively or negatively affect expression from even the strongest enhancers. Principal component analysis (PCA) indicates that enhancer and promoter function can be explained by a single principal component while UTR function involves multiple functional components. The PCA also highlights outliers and suggest differences in mechanisms of regulation by individual elements. Our data also identify numerous regulatory cassettes composed of different individual regulatory elements that exhibit equivalent gene expression levels. These data thus provide a catalog of elements that could in future be used in the design of synthetic regulatory circuits

From Structural Variation of Gene Molecules to Chromatin Dynamics and Transcriptional Bursting

Transcriptional activation of eukaryotic genes is accompanied, in general, by a change in the sensitivity of promoter chromatin to endonucleases. The structural basis of this alteration has remained elusive for decades; but the change has been viewed as a transformation of one structure into another, from “closed” to “open” chromatin. In contradistinction to this static and deterministic view of the problem, a dynamical and probabilistic theory of promoter chromatin has emerged as its solution. This theory, which we review here, explains observed variation in promoter chromatin structure at the level of single gene molecules and provides a molecular basis for random bursting in transcription—the conjecture that promoters stochastically transition between transcriptionally conducive and inconducive states. The mechanism of transcriptional regulation may be understood only in probabilistic terms