The eukaryotic genome is packaged by wrapping ~147 bp units of DNA around histone octamers to form chains of nucleosomes. The packaging of the DNA within a nucleosome reduces access of the DNA to most transcription factors and polymerases. In between nucleosomes, there are regions of more accessible DNA, called linker regions that vary from a few base pairs to several hundred base pairs. Thus within any given cell type, the precise partitioning of the genome into nucleosome-bound and nucleosome-free DNA regions can have large consequences on gene regulation and help define a particular cellular state. Recent studies suggest that the genome plays a large role in encoding its own packaging through differences in affinity of the underlying sequence for the histone octamer. Nucleosome locations are also regulated by several different ATP-dependent chromatin remodeling enzymes, which enable rapid rearrangements in chromatin structure in response to developmental cues. Thirdly, the in vivo nucleosome assembly process involves proteins called histone chaperones. No individual factor is capable of playing a dominant role in generating the immense specificity required to regulate transcription in eukaryotes. This gives rise to the question of what are the relative contributions to nucleosome positions due to each of these factors. This question has been investigated with biochemical reconstitutions and activity assays, which tracked nucleosome position distributions and kinetics in the presence of various factors. Our data support a model in which remodeling enzymes move nucleosomes to new locations by a general sequence-independent mechanism. However, consequent to the rate-limiting remodeling step, the local DNA sequence promotes a collapse of remodeling intermediates into highly resolved positions that are dictated by thermodynamic differences between adjacent positions. Analogously, histone chaperones have been found to reduce thermodynamic equilibration among all available nucleosome positions, but leads to local equilibration after a rapid histone deposition step. Future understanding of how these factors coordinate their activities in vivo and in the presence of transcription factors, will hopefully lead to better predictive models of gene regulation.

G protein-coupled receptor (GPCR) signal transduction is a fundamental building block of cell biology, yet how different ligands such as peptide agonists and small molecules can act at the same receptor to produce different functional outcomes is not fully appreciated. A proliferation of biochemical, biophysical, and pharmacological studies led to an abundance of receptor active-state structures and candidate molecules selective for desired outcomes while avoiding side effects. With preponderance of data, however, also came contradictions and challenges in interpretations. This thesis returns to the basic step of ligand-receptor-effector interaction, and explores two fundamental questions: 1) can different agonists selectively couple the receptor to different cytoplasmic proteins in intact cells? and 2) in the agonist-selective receptor endocytosis pathway, how do GPCR kinases (GRKs) recognize different agonist-bound receptors? Using two engineered protein biosensors as discreet intracellular proteins, two agonists showed distinctly different profiles in recruiting the protein biosensors. This result provides a proof of concept of a potential answer to the first question, that in a ‘clean’ cellular environment without native signaling interference, agonists can indeed engage two different biosensors with selective preferences. Similarly for the second question, using an engineered GRK-free cellular background, a single GRK subtype was shown to produce agonist-selective functional outcome of receptor internalization. This GRK was also recruited to the receptor in an agonist-selective manner, supporting the hypothesis that selective functional outcomes begin upstream at selective receptor-GRK interactions. Furthermore, a single domain was shown to be the key selectivity determinant for GRK recruitment to the receptor and internalization function.

Long-term silencing of large regions of the genome is achieved through the formation of heterochromatin. From yeast to humans, heterochromatin is characterized by two key molecular signatures: (i) di or tri-methylation of lysine 9 of histone H3 (H3K9me2/3), and (ii) heterochromatin protein 1 (HP1). The association of HP1 with H3K9-methylated chromatin drives heterochromatin assembly and spread. Yet, how HP1 assembles on methylated nucleosomal templates and how the HP1-nucleosome complex is regulated are poorly understood.

Using S. pombe as a model system, we show that two dimers of the HP1 protein, Swi6, binds to one nucleosome: each dimer contains one chromodomain (CD) that engages one copy of the H3K9-methyl mark, while the other CD is unoccupied. This HP1-nucleosome complex acts as a scaffold for the addition of other HP1 molecules that self-associate through a novel CD-CD interface nucleating from the unoccupied CDs. Chromodomain-mediated polymerization of HP1 on chromatin appears to (1) increase its association with methylated nucleosomes in vitro, (2) bridge neighboring methylated nucleosomes, and (3) increase heterochromatin assembly in vivo.

Our data suggests that H3K9-methyl recognition and chromatin coating by HP1 are intrinsic to the fundamental architecture of the HP1-nucleosome complex. But they also raise the question of how methylated chromatin templates HP1 assembly.

We found that two key features of heterochromatin, the H3K9me3 and the nucleosomal DNA, promote a conformational change in Swi6 that drives its association with nucleosomes. By binding to methylated nucleosomes, unbound Swi6 dimers switch from an autoinhibited state that is refractory to both methyl mark recognition and higher-order oligomerization to a state that is competent for spreading. Cryo-EM studies of the Swi6-nucleosome complex reveal the architecture of the spreading competent state. In vivo, mutants that disrupt such a switch also result in disruption of heterochromatin.

The coupling of a conformational switch in HP1 to the recognition of specific features of methylated chromatin provides a mechanism for how HP1 can specifically target H3K9-methylated chromatin, thus preventing its aberrant spread into euchromatin. Finally, our discovery of these different HP1 conformational states provides a basic starting point for understanding how HP1 can switch between alternative functions in heterochromatin.

DNA methylation and histone H3 lysine 9 methylation (H3K9me) are hallmarks of heterochromatin in plants and mammals and are successfully maintained across generations. The biochemical and structural basis for this maintenance is poorly understood. The maintenance DNA methyltransferase from Zea mays, ZMET2, recognizes H3K9me2 via a chromodomain (CD) and bromo-adjacent homology domain (BAH), which flank the catalytic domain. Here, we show that the H3K9me mark allosterically stimulates ZMET2 activity and also substantially increases the specificity of ZMET2 for hemimethylated versus unmethylated DNA. Interestingly, dinucleosomes are the preferred ZMET2 substrate, with DNA methylation targeted primarily to linker DNA. Further, while the CD stabilizes ZMET2 binding, the BAH domain is critical for catalysis and serves as an allosteric regulator of the enzyme. Negative stain electron microscopy shows a single ZMET2 molecule bridging two nucleosomes within a dinucleosome, consistent with the biochemical analyses. We propose a model in which CD-mediated binding and allosteric activation via BAH/H3K9me interactions coupled with nucleosome bridging allows ZMET2 to correctly read the chromatin context and thereby faithfully maintain DNA methylation and reinforce heterochromatic elements across the genome.

Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands\cite{pmid21346195,pmid10753776,pmid9169472}. The mechanism of how HP1 achieves this silencing activity is not yet described, though much work has shown it associates with many diverse chromatin components and on many different timescales. We identified a new property of the human HP1$\alpha$ protein: the ability to form phase-separated droplets. While unmodified HP1$\alpha$ is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation driven phase separation can be promoted or reversed by specific HP1$\alpha$ ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1$\alpha$ droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we found that both unmodified and phosphorylated HP1$\alpha$ induce rapid compaction of DNA strands into puncta, although with different characteristics\cite{pmid20580955}. By direct protein delivery into mammalian cells we found that an HP1$\alpha$ mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1$\alpha$. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context. Importantly, this activity is rooted in mechanistic changes within the HP1 protein itself. The ability to assemble and phase separate is prefaced by a large conformational change directed through the unstructured NTE, CTE, and hinge regions. The change is dependent on both length and composition of charges/hydrophobicity within these regions. This leads to a belief that though rooted in regions that appear unstructured, there is delicate mechanism behind the ordered transition to a phase-separated assembly.

In my dissertation, I present a comprehensive investigation into chitinase enzymes and their diverse roles in biology, as well as contributions to antiviral drug development and social justice initiatives. The first chapter focuses on Acidic Mammalian Chitinase (AMCase), an enzyme responsible for degrading the resilient polysaccharide chitin in mammalian stomachs and lungs. Through the development of novel chitinase activity assays, we explore the effects of asthma-associated mutations, examine the contributions of individual enzyme domains in degrading crystalline chitin, and compare the behavior of AMCase with chitotriosidase, another chitinase in mammals. Additionally, we explore the challenges of engineering hyperactive chitinases, highlighting the limitations of traditional screening methods when assessing complex chitin substrates.In the second chapter, I focus on the SARS-CoV-2 macrodomain (Mac1), a viral protein that antagonizes host antiviral signaling. Using computational and structural techniques, we identify numerous small molecules that bind to the active site of the macrodomain, providing a foundation for the development of potent SARS-CoV-2 macrodomain inhibitors. We validate these findings through solution binding assays, utilizing multiple biophysical techniques to confirm fragment hits. In the third chapter, I delve into the catalytic mechanism underlying the pH-dependent activity profile of mouse Acidic Mammalian Chitinase (mAMCase). By employing a combination of biochemical, structural, and computational modeling approaches, I uncovered the ability of the mouse homolog to function effectively in both acidic and neutral environments. I determined the kinetic properties of mAMCase across a broad pH range and reveal its intriguing dual activity optima at pH 2 and 7. Through high-resolution crystal structures of mAMCase bound to chitin, I unveiled extensive conformational ligand heterogeneity, providing valuable insights into the catalytic mechanism of mAMCase. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. In the fourth chapter, I outline my contributions to advancing diversity, equity, inclusion, and justice (DEIJ) at UCSF, other academic institutions, and beyond academia. This chapter not only recounts my experiences cultivating LGBTQ+ community, advocating for racial justice, and interrogating institutional policies regarding graduate admissions, but also renders visible otherwise neglected contributions to improving the social conditions in which we perform science.

RSC, an essential chromatin remodeling complex in budding yeast, is involved in a variety of biological processes including transcription, recombination, repair and replication. How RSC participates in such diverse processes is not fully understood. In vitro, RSC uses ATP to carry out several seemingly distinct reactions: it repositions nucleosomes, transfers H2A/H2B dimers between nucleosomes and transfers histone octamers between pieces of DNA. This raises the intriguing mechanistic question of how this molecular machine can use a single ATPase subunit to create these varied products. Here, we use a FRET-based approach to kinetically order the products of the RSC reaction. Surprisingly, transfer of H2A/H2B dimers and histone octamers is initiated on a time scale of seconds when assayed by FRET, but formation of stable nucleosomal products occurs on a time scale of minutes when assayed by native gel. These results suggest a model in which RSC action rapidly generates an unstable encounter intermediate that contains the two exchange substrates in close proximity. This intermediate then collapses more slowly to form the stable transfer products seen on native gels. The rapid, biologically relevant time scale on which the transfer products are generated implies that such products can play key roles in vivo.

Chromatin structure regulates the accessibility of DNA in several crucial nuclear processes, including transcription, replication and recombination. The ISWI-family of chromatin remodeling complexes translationally repositions histone octamers on DNA. One member of this family, the ATP-dependent chromatin assembly factor (ACF), generates regularly spaced nucleosomes in vitro and is required for transcriptional silencing in vivo.

To dissect ACF mechanism, we used a FRET based approach to follow the movement of DNA with respect to the histone octamer in real time. We find that ACF generates at least one transient intermediate in which part of the DNA has moved across the histone octamer. The rate of ACF remodeling is sensitive to the length of the linker DNA such that shortening the linker DNA below 60 bp progressively slows down both ATP hydrolysis and remodeling. As a result, ACF generates and maintains nucleosome spacing by constantly moving nucleosomes towards longer flanking DNA faster than shorter flanking DNA. ACF thus creates a dynamic equilibrium between different nucleosome positions such that the centered position is most highly populated.

To understand how ACF can rapidly sample both sides of a nucleosome to accomplish bidirectional movement, my collaborators and I employed a myriad of techniques to conclude that ACF functions as a dimeric molecular motor. We found that ACF binds nucleosomes in a cooperative manner, and that this oligomeric state is required for back and forth remodeling of the nucleosome. Additionally, the ATPase subunit energetically couples different nucleotide states with distinct reaction intermediates, and thus we observe that the physical interactions with the nucleosome change as a function of the ATP reaction cycle. Lastly, we found that ACF utilizes ATP in three distinct ways, and that translocation is characterized by distinct pauses during movement of DNA relative to the histone octamer.

In summary, these data provide a mechanistic basis for the directionality of nucleosome repositioning and explain the centering activity of ACF. Futhermore, the dimeric architecture of ACF allows for the rapid and processive bidirectional nucleosome movement required for nucleosome spacing.