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Open Access Publications from the University of California

Scholarly Works (8 results)

  • Article
  • Peer Reviewed
UC Santa Cruz Previously Published Works (2020)
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.
    Cover page: Nucleosomal proofreading of activator–promoter interactions
    • Thesis
    • Peer Reviewed
    UC Santa Cruz Electronic Theses and Dissertations (2020)

    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.

      Cover page: Specificity in Transcriptional RegulationCreative Commons 'BY-NC-ND' version 4.0 license
      • Article
      • Peer Reviewed
      UC Santa Cruz Previously Published Works (2015)
      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.
        Cover page: From Structural Variation of Gene Molecules to Chromatin Dynamics and Transcriptional Bursting
        • Article
        • Peer Reviewed
        UC Santa Cruz Previously Published Works (2020)
        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.
          Cover page: Permutational analysis of Saccharomyces cerevisiae regulatory elements
          • Article
          • Peer Reviewed
          UC Santa Cruz Previously Published Works (2024)
          Fluctuations in the initiation rate of transcription, the first step in gene expression, ensue from the stochastic behavior of the molecular process that controls transcription. In steady state, the regulatory process is often assumed to operate reversibly, i.e., in equilibrium. However, reversibility imposes fundamental limits to information processing. For instance, the assumption of equilibrium is difficult to square with the precision with which the regulatory process executes its task in eukaryotes. Here we provide evidence - from microscopic analyses of the transcription dynamics at a single gene copy of yeast - that the regulatory process for transcription is cyclic and irreversible (out of equilibrium). The necessary coupling to reservoirs of free energy occurs via sequence-specific transcriptional activators and the recruitment, in part, of ATP-dependent chromatin remodelers. Our findings may help explain how eukaryotic cells reconcile the dual but opposing requirements for fast regulatory kinetics and high regulatory specificity.
            Cover page: Single gene analysis in yeast suggests nonequilibrium regulatory dynamics for transcription.
            • Article
            • Peer Reviewed
            UC Santa Cruz Previously Published Works (2019)
            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.
              Cover page: Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome
              • Article
              • Peer Reviewed
              UC Santa Cruz Previously Published Works (2022)
              The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene expression.
                Cover page: The MuvB complex binds and stabilizes nucleosomes downstream of the transcription start site of cell-cycle dependent genes
                • Article
                • Peer Reviewed
                UC Santa Cruz Previously Published Works (2024)
                Genome-wide identification of chromatin organization and structure has been generally probed by measuring accessibility of the underlying DNA to nucleases or methyltransferases. These methods either only observe the positioning of a single nucleosome or rely on large enzymes to modify or cleave the DNA. We developed adduct sequencing (Add-seq), a method to probe chromatin accessibility by treating chromatin with the small molecule angelicin, which preferentially intercalates into DNA not bound to core nucleosomes. We show that Nanopore sequencing of the angelicin-modified DNA is possible and allows visualization and analysis of long single molecules with distinct chromatin structure. The angelicin modification can be detected from the Nanopore current signal data using a neural network model trained on unmodified and modified chromatin-free DNA. Applying Add-seq to Saccharomyces cerevisiae nuclei, we identified expected patterns of accessibility around annotated gene loci in yeast. We also identify individual clusters of single molecule reads displaying different chromatin structure at specific yeast loci, which demonstrates heterogeneity in the chromatin structure of the yeast population. Thus, using Add-seq, we are able to profile DNA accessibility in the yeast genome across long molecules.
                  Cover page: Probing chromatin accessibility with small molecule DNA intercalation and nanopore sequencing
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