Our laboratory investigates the function of chromatin - specifically the roles of histone modifications - in regulation of specific biological processes and their alteration in human disease such as cancer using biochemical, genetic, molecular biological and high-throughput approaches. While 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 histone modifications. We seek and welcome collaborations that will enrich and enhance our research by expanding our 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 void of hyperbole and cynicism. As students of life sciences, we are enamored by the biology of our genome and feel privileged to explore the perplexity of its structure for the pure joy of discovery and the betterment of society.
Histone acetylation is thought to function locally at specific genomic loci to regulate various DNA-based processes including gene expression. Numerous alterations in locus-specific patterns of histone acetylation have been reported in cancer but none has been related correlatively or causally to clinical outcome. In addition to local regulation, histone acetylation can also be modulated at a global level, as assessed by, for instance, immunehistochemical examination of primary tissues. Our laboratory has discovered that cancer tissues with lower global levels of histone acetylation display significantly increased rate of tumor recurrence or cancer-related mortality, findings that have been validated independently. However, the function of global changes in histone acetylation in normal biology and how it might contribute to the cancer phenotype have been completely unknown and unexplainable by the current mechanistic paradigms.
Chromatin has been thought to regulate only DNA-based processes. However, we have discovered that global and dynamic acetylation and deacetylation of histones is in constant flux with exogenous acetate, and that chromatin regulates the direction of this flux in response to intracellular pH (pHi) alterations. In acidic conditions, histones are globally deacetylated and the resulting acetate molecules are co-transported with protons out of the cell through the monocarboxylate transporters (MCTs), thereby decreasing the intracellular proton load. At high pH, histones are globally acetylated, serving to store acetate molecules and resisting further increases in pHi. This process requires histone deacetylase (HDAC) activity, inhibition of which lowers intracellular pH, providing a novel mechanism of action for HDAC inhibitors. Based on these data, we have proposed that acetylation of chromatin functions as a rheostat to regulate pHi. These findings have also provided a plausible hypothesis that cancer cells with lower levels of histone acetylation may be experiencing an acidic microenvironment and are actively deacetylating their histones and extruding acetate and protons to maintain their intracellular pH. This process could occur in different types of cancer and also in normal cells within cancer tissues that are subjected to the same level of acidity.
We now aim to determine how levels and distribution of histone acetylation throughout chromatin integrates response to pH with gene expression and cellular phenotype and the mechanisms underlying such integration. We would like to determine how HATs and HDACs may sense changes in pH and modulate their level of enzymatic activity to balance the global acetylation and deacetylation in favor one or the other. We expect this line of inquiry to uncover other links between histone modifications and specific cellular physiological and metabolic processes.
Viral oncoproteins have provided useful tools for investigating several aspects of cancer cell biology. DNA viruses often initiate infection of host animals by infecting cells that are fully differentiated, and have left the cell cycle. Such non-cycling cells are poor hosts for viral replication. Consequently, many of these viruses have evolved proteins, expressed immediately after infection, that force the host cell back into the cell cycle, inducing expression of the cellular biosynthetic machinery required for producing progeny virions. In doing so, viruses have become invaluable tools for uncovering central molecular processes that regulate cell proliferation such as the functions of retinoblastoma (Rb) tumor suppressor, and its family members p130 and p107, in controlling the cell cycle, and the importance of p53 inactivation in tumorigenesis.
The DNA tumor virus Adenovirus expresses the E1A and E1B proteins that can transform mammalian cells to an oncogenic phenotype. A splicing variant of E1A named small e1a, which contains three of the four conserved regions (CR1, 2 and 4) among primate adenovirus E1As, retains most of the oncogenic potential of this virus. Small e1a consists of 243 amino acids and has the ability to force cell cycle-arrested primary human fibroblasts into S-phase. In our laboratory, we have discovered that through dynamic and temporal binding to the host genome, e1a orchestrates a precise re-arrangement of multiple regulators of gene expression including those with epigenetic activities in the course of S-phase induction in cells that should remain in a resting state—a fundamental feature of cancer cells. In effect, e1a has provided us with an epigenetic blueprint for cellular transformation. Among the numerous histone modifications, e1a specifically targets H3K18ac for an “epigenetic makeover”, erasing most of the peaks across the genome including from enhancers of cell-type specific genes and establishing new ones mainly at promoters of genes that function in cell cycle and are normally repressed by Rb. The strategic redistribution of H3K18ac by e1a highlights the importance of this modification for transcriptional control, a phenomenon that we are currently trying to understand at a mechanistic level. Overall, our data have underscored the cellular transformation by adenovirus e1a as a powerful system to study the epigenetic changes that occur during an oncogenic reprogramming process. This system is reproducible, manipulatable and amenable to temporal measurements with proven relevance to primary human cancer.
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.