Research

RESEARCH OUTLOOK

Our laboratory investigates the functions of histones in regulation of specific biological processes and their alterations in human disease using biochemical, genetic, molecular biological and high-throughput approaches. Although we are agnostic to model systems, we choose and employ one that is most appropriate for the question being asked. Presently, we use cancer cell lines, human and murine embryonic stem cells, primary human cancer tissues as well as the model organism, Saccharomyces cerevisiae, to study the basic biology and pathophysiology of histones. We seek and welcome collaborations that will mutually enrich and enhance research by expanding technical and intellectual repertoire. Our mission is to cultivate a culture of intellectual curiosity and experimental exploration unencumbered by perceived dogma. We take deliberate risks to enable inquiry into uncharted territories, yet practice within our scientific space in order to incur the funds necessary to move forward with our unique vision and speculations. We encourage imaginative, inspired and original thinking that produce new scientific narratives devoid of hyperbole and cynicism. As students of life sciences, we are enamored by the biology of chromatin and feel privileged to explore the perplexity of its structure for the pure joy of discovery and the betterment of society.

CURRENT PROJECTS

I. Understanding Histones and Chromatin vis-à-vis Histone H3 Copper Reductase Activity

Histones were initially assumed to mainly enable the packaging of large amounts of eukaryotic DNA into the confines of the nucleus. Pioneering experiments in the 1980s and 1990s revealed that histones also function in regulating gene expression and essentially all other DNA-based processes (recognized in 2018 by Lasker Awards to Grunstein and Allis). However, ancestral histones were present in organisms with small genomes, no nucleus and little ability for epigenetic regulation, suggesting that histones may have an additional, unknown function that served as the original impetus for their evolution. We have indeed discovered a novel function for the histone H3-H4 tetramer, the structure most similar to ancestral histone complexes. H3-H4 tetramer bound to DNA docks with two H2A-H2B dimers to form the canonical 147-bp eukaryotic nucleosome. Amino acid residues in the C-terminal regions of the two opposing histone H3 proteins, including cysteine 110 (H3C110) and histidine 113 (H3H113), form a pocket highly reminiscent of known copper binding sites but with no previously recognized functional significance. We surmised that this region may not only bind Cu2+ but serve as a catalytic site for its reduction to Cu1+. To determine whether histones enzymatically affect the oxidation state of copper, we have developed a novel assay for copper reduction and shown that the H3-H4 tetramer assembled from recombinant histones with no modifications binds Cu2+ with high affinity and catalyzes its reduction to Cu1+. We have also demonstrated that perturbation of the active site affects Cu1+ levels and copper-dependent activities in vivo. We have developed a reporter system based on an endogenous transcription factor that directly senses Cu1+ levels and modulates target gene expression accordingly. The in vivo reporter indicated that loss- and gain-of-function mutations of the H3 active site decrease and increase intracellular Cu1+ levels, respectively. These mutations had the expected functional effects on mitochondrial ETC as well as on Cu, Zn-superoxide dismutase 1 (Sod1), a copper-dependent enzyme involved in oxidation defense. Altogether, we have provided extensive evidence that the histone H3-H4 tetramer is the first known nucleocytoplasmic copper reductase in any organism, establishing a new paradigm for understanding chromatin structure and function as an enzyme. As the emergence of eukaryotes coincided with the Great Oxidation Event and decreased biousability of metals, we have proposed that the H3 enzymatic function may have facilitated eukaryogenesis by contributing vital Cu1+ to protomitochondria. Our future aims are broadly as follows (in no particular order):

  • To understand the mechanism of catalysis and the contributions of the residues in and around the active site.
  • To discern how the activity is regulated through post-translational modifications, specific sequence features of histones and histone variants, and in the context of nucleosomes.
  • To identify the genes and pathways that integrate the enzymatic activity of histones with cellular copper homeostasis.
  • Identify and characterize the molecular factors that link histone enzyme activity to mitochondria, a major consumer of copper.
  • The role of histone enzyme activity on cellular and mitochondrial iron homeostasis as iron uptake and mobilization depends on copper.

II. Linking the Novel Histone Enzyme Activity to Human Disease

We are also interested in understanding how the enzymatic function of histone H3 may contribute to human disease. As a bioessential metal, copper is required for enzymes in diverse biological functions such as respiration (cytochrome c oxidase), antioxidant activity (Sod1), iron homeostasis (multicopper oxidases), tissue integrity (lysyl oxidase), methylation cycle (methionine synthase), melanin synthesis (tyrosinase), and neurotransmitter synthesis (dopamine-β-hydroxylase). Copper serves as a co-factor in Cu2+ or Cu1+ oxidation states. However, it is the Cu1+ ions that are trafficked intracellularly, indicating a need to maintain copper in its reduced form for proper cellular distribution. Copper is also an important but overlooked modulator of cell signaling. Our discovery of histone copper reductase activity has established the first protein-based mechanism for modulating the redox state of copper and thus copper toxicity. This is because the reduced form of copper ions (Cu1+ or cuprous ions), not its cupric form (Cu2+), causes the most harmful effects of copper including protein mis-metallation and oxidative damage. Therefore, the optimal levels of Cu1+ inside the cell must be maintained within a ‘Goldilocks zone’—too little Cu1+ would impair important processes, and too much would be toxic. Furthermore, the fact that the histone enzyme activity consumes reducing equivalents (e.g., NADPH), a limiting nutrient for metabolically active or proliferating cells, adds an additional layer of intricacy to regulation of Cu1+ ion production. Thus histone enzyme activity may be impacted by the redox state of the cell as it balances Cu1+ production, consumption of reducing equivalents, and copper toxicity. We would like to reveal the contribution of histone enzyme activity to human diseases such as cancer and certain neurodegenerative disorders in which copper dysfunction has been implicated; investigate the effects of histone cancer mutations on enzyme activity; and identify and develop small molecules inhibitors of histone enzyme activity to establish proof-of principle therapeutic approaches based on specifically thwarting toxicity of Cu1+. Our findings hold great potential to reveal an entirely unexplored but fundamental molecular regulatory axis with major therapeutic implications for a large class of human diseases.

III.  Evolution of Genome Compaction and its Biological Consequences

Evolution of eukaryotes from single-cell to multicellular species has been associated with considerable expansion of genome size—defined as the haploid content of DNA.  However, nuclear volume has not increased proportionally to genome size, requiring organisms with larger genomes to compact their chromatin to greater extents than organisms with smaller genomes. These observations have raised the fundamental question of how eukaryotes manage to compact the genome to different degrees depending on the ratio of genome size to nuclear volume. Although higher eukaryotes have developed evolved specific mechanisms for genome compaction such as linker histones, it has not been known whether histones themselves have evolved to also facilitate greater compaction in species with large genomes. Therefore we asked whether any of the histone proteins have co-evolved with genome size to regulate genome compaction. An extensive computational analysis of the canonical histone sequences in 160 fully sequenced eukaryotic genomes spanning the eukaryotic kingdom including protozoa, fungi, plants and animals revealed that the histone H2A N-terminal domain (NTD) has systematically co-evolved with the expansion of genome size by acquiring positively charged arginine residues and losing serine/threonine residues at specific positions. Using genetic, molecular and in vitro biochemical approaches, we have demonstrated that the evolutionary changes in H2A directly and significantly regulate chromatin compaction.  Insertion of specific residues that appear in larger genomes into the H2A of budding yeast, a small genome organism, causes chromatin to compact by as much as 40%!  Conversely, removal of evolutionary residues in H2A de-compacts human chromatin so substantially that it is clearly visible in micrographs and leads to increased nuclear size.  Interestingly, changes in chromatin compaction affect nuclear volume, with increased chromatin compaction leading to decreased nuclear volume.  Our findings have revealed a novel and simple evolutionary principle for regulation of genome compaction across eukaryotes through a previously unsuspected region of the nucleosome.

Our findings have now raised the question of what molecular processes govern the optimal levels of genome compaction and nuclear volume. If genomes can be more or less compact, why aren’t they? This issue pertains not only to our fundamental knowledge but also to human disease including cancer, a hallmark of which is aberrant nuclear morphology due to deregulated chromatin compaction. In fact, over 70% of reported cancer-related mutations in the H2A NTD involve the evolutionary residues that we have found to regulate chromatin compaction. Our efforts are now focused on understanding the evolutionary logic of genome compaction through canonical histones and to discover the molecular processes that are compromised by altered genome compaction.