Faculty Bibliography

H3K56 acetylation (H3K56Ac) was first identified in yeast and has recently been reported to play important roles in maintaining genomic stability, chromatin assembly, DNA replication, cell cycle progression and DNA repair. Although H3.1K56Ac has been relatively well studied, the function of H3.3K56Ac remains mostly unknown in mammals. In this study, we used H3.3K56Q and H3.3K56R mutants to study the possible function of H3.3K56 acetylation. The K-to-Q substitution mimics a constitutively acetylated lysine, while the K-to-R replacement mimics a constitutively unmodified lysine. We report that cell lines harbouring mutation of H3.3K56R exhibit increased cell death and dramatic morphology changes. Using a Tet-Off inducible system, we found an increased population of polyploid/aneuploid cells and decreased cell viability in H3.3K56R mutant cells. Consistent with these results, the H3.3K56R mutant had compromised H3.3 incorporation into several pericentric and centric heterochromatin regions we tested. Moreover, mass spectrometry analysis coupled with label-free quantification revealed that biological processes regulated by the H3.3-associating proteins, whose interaction with H3.3 was markedly increased by H3.3K56Q mutation but decreased by H3.3K56R mutation, include sister chromatid cohesion, mitotic nuclear division, and mitotic nuclear envelope disassembly. These results suggest that H3.3K56 acetylation is crucial for chromosome segregation and cell division in mammals.

Nickel (Ni) is carcinogenic to humans, and causes cancers of the lung, nasal cavity, and paranasal sinuses. The primary mechanisms of Ni‑mediated carcinogenesis involve the epigenetic reprogramming of cells and the ability for Ni to mimic hypoxia. However, the exact mechanisms of carcinogenesis related to Ni are obscure. Nuclear protein 1 (NUPR1) is a stress‑response gene overexpressed in cancers, and is capable of conferring chemotherapeutic resistance. Likewise, activator protein 1 (AP‑1) is highly responsive to environmental signals, and has been associated with cancer development. In this study, NUPR1 was found to be rapidly and highly induced in human bronchial epithelial (BEAS‑2B) cells exposed to Ni, and was overexpressed in Ni‑transformed BEAS‑2B cells. Similarly, AP‑1 subunits, JUN and FOS, were induced in BEAS‑2B cells following Ni exposure. Knockdown of JUN or FOS was found to significantly suppress NUPR1 induction following Ni exposure, demonstrating their importance in NUPR1 transactivation. Reactive oxygen species (ROS) are known to induce AP‑1, and Ni has been shown to produce ROS. Treatment of BEAS‑2B cells with antioxidants was unable to prevent NUPR1 induction by Ni, suggesting that NUPR1 induction by Ni relies on mechanisms other than oxidative stress. To determine how NUPR1 is transcriptionally regulated following Ni exposure, the NUPR1 promoter was cloned and inserted into a luciferase gene reporter vector. Multiple JUN binding sites reside within the NUPR1 promoter, and upon deleting a JUN binding site in the upstream most region within the NUPR1 promoter using site‑directed mutagenesis, NUPR1 promoter activity was significantly reduced. This suggests that AP‑1 transcriptionally regulates NUPR1. Moreover, knockdown of NUPR1 significantly reduced colony formation and anchorage‑independent growth in Ni‑transformed BEAS‑2B cells. Therefore, these results collectively demonstrate a novel mechanism of NUPR1 induction following Ni exposure, and provide a molecular basis by which NUPR1 may contribute to lung carcinogenesis.

Replication-dependent canonical histone messenger RNAs (mRNAs) do not terminate with a poly(A) tail at the 3' end. We previously demonstrated that exposure to arsenic, an environmental carcinogen, induces polyadenylation of canonical histone H3.1 mRNA, causing transformation of human cells in vitro. Here we report that polyadenylation of H3.1 mRNA increases H3.1 protein, resulting in displacement of histone variant H3.3 at active promoters, enhancers, and insulator regions, leading to transcriptional deregulation, G2/M cell-cycle arrest, chromosome aneuploidy, and aberrations. In support of these observations, knocking down the expression of H3.3 induced cell transformation, whereas ectopic expression of H3.3 attenuated arsenic-induced cell transformation. Notably, arsenic exposure also resulted in displacement of H3.3 from active promoters, enhancers, and insulator regions. These data suggest that H3.3 displacement might be central to carcinogenesis caused by polyadenylation of H3.1 mRNA upon arsenic exposure. Our findings illustrate the importance of proper histone stoichiometry in maintaining genome integrity.

OBJECTIVES/OBJECTIVE:Importin-4 (IPO4) is responsible for transporting histones H3 and H4 into the nucleus for chromatin assembly. But, the role of IPO4 in cancer, especially in gastric cancer (GC), has not been fully understood. We aim to determine the expression and function of IPO4 in GC. MATERIALS AND METHODS/METHODS:Bioinformatics analysis was used to study the association of IPO4 and GC using GEO data and the Kaplan-Meier plotter. The quantitative real-time polymerase chain reaction and Western blot analysis were used to determine the IPO4 level in GC cells and tissues. Small interfering RNAs (siRNAs) were used to knockdown endogenous IPO4 expression in GC cells. Cell counting kit-8 (CCK-8), colony formation and transwell assays were used to examine the effect of IPO4 on cell proliferation and migration. RESULTS:IPO4 mRNA is overexpressed in GC tissues using bioinformatics analysis of three groups' transcriptome data, and high level of IPO4 is negatively correlated with poor long-term survival using the Kaplan-Meier plotter analysis. Western blot analysis further shows that IPO4 protein levels are also overexpressed in GC tissues and a number of GC cell lines. Endogenous IPO4 level can be inhibited by specific siRNA effectively. Importantly, CCK-8, colony formation, and transwell assays demonstrate that IPO4 knockdown by siRNA impairs GC cell proliferation and migration. CONCLUSIONS:Our data suggest that IPO4 contributes to GC progression and poor prognosis, and may function as a driving force in GC progression.

Alzheimer's disease (AD) is the most common form of dementia. The accumulation of β-amyloid plaques and intracellular neurofibrillary tangles of hyperphosphorylated tau protein are two hallmarks of AD. The β-amyloid and tau proteins have been at the center of AD research and drug development for decades. However, most of the clinical trials targeting β-amyloid have failed. Whereas the safety and efficacy of most tau-targeting drugs have not yet been completely assessed, the first tau aggregation inhibitor, LMTX, failed in a late-stage trial, leading to further recognition of the complexities of AD and reconsideration of the amyloid hypothesis and perhaps the tau hypothesis as well. Multilevel complex interactions between genetic, epigenetic, and environmental factors contribute to the occurrence and progression of AD. Formaldehyde (FA) is a widespread environmental organic pollutant. It is also an endogenous metabolite in the human body. Recent studies suggest that elevation of FA in the body by endogenous and/or exogenous exposure may play important roles in AD development. We have demonstrated that FA reduces lysine acetylation of cytosolic histones, thereby compromising chromatin assembly and resulting in the loss of histone content in chromatin, a conserved feature of aging from yeast to humans. Aging is an important factor for AD progression. Therefore, FA-induced inhibition of chromatin assembly and the loss of histones may contribute to AD initiation and/or development. This review will briefly summarize current knowledge on mechanistic insights into AD, focusing on epigenetic alterations and the involvement of FA in AD development. The exploration of chemical exposures as contributing factors to AD may provide new insights into AD mechanisms and could identify potential novel therapeutic targets.

Environmental stress such as genotoxic agents can cause DNA damage either indirectly through the generation of reactive oxygen species or directly by interactions with the DNA molecule. Damage to the genetic material may cause mutations and ultimately cancer. Genotoxic mutation can be prevented either by apoptosis or DNA repair. In response to DNA damage, cells have evolved DNA damage responses (DDR) to detect, signal, and repair DNA lesions. Epigenetic mechanisms play critically important roles in DDR, which requires changes in chromatin structure and dynamics to modulate DNA accessibility. Incorporation of histone variants into chromatin is considered as an epigenetic mechanism. Canonical histones can be replaced with variant histones that change chromatin structure, stability, and dynamics. Recent studies have demonstrated involvement of nearly all histone variants in environmental-stress-induced DNA damage repair through various mechanisms, including affecting nucleosome dynamics, carrying variant-specific modification, promoting transcriptional competence or silencing, mediating rearrangement of chromosomes, attracting specific repair proteins, among others. In this review, we will focus on the role of histone variants in DNA damage repair after exposure to environmental genotoxic agents. Understanding the mechanisms regulating environmental exposure-induced epigenetic changes, including replacement of canonical histones with histone variants, will promote the development of strategies to prevent or reverse these changes.

As the primary metabolite of alcohol and the most abundant carcinogen in tobacco smoke, acetaldehyde is linked to a number of human diseases associated with chronic alcohol consumption and smoking including cancers. In addition to direct DNA damage as a result of the formation of acetaldehyde-DNA adducts, acetaldehyde may also indirectly impact proper genome function through the formation of protein adducts. Histone proteins are the major component of the chromatin. Post-translational histone modifications (PTMs) are critically important for the maintenance of genetic and epigenetic stability. However, little is known about how acetaldehyde-histone adducts affect histone modifications and chromatin structure. The results of protein carbonyl assays suggest that acetaldehyde forms adducts with histone proteins in human bronchial epithelial BEAS-2B cells. The level of acetylation for N-terminal tails of cytosolic histones H3 and H4, an important modification for histone nuclear import and chromatin assembly, is significantly downregulated following acetaldehyde exposure in BEAS-2B cells, possibly due to the formation of histone adducts and/or the decrease in the expression of histone acetyltransferases. Notably, the level of nucleosomal histones in the chromatin fraction and at most of the genomic loci we tested are low in acetaldehyde-treated cells as compared with the control cells, which is suggestive of inhibition of chromatin assembly. Moreover, acetaldehyde exposure perturbs chromatin structure as evidenced by the increase in general chromatin accessibility and the decrease in nucleosome occupancy at genomic loci following acetaldehyde treatment. Our results indicate that regulation of histone modifications and chromatin accessibility may play important roles in acetaldehyde-induced pathogenesis. Environ. Mol. Mutagen., 2018. © 2018 Wiley Periodicals, Inc.

[This corrects the article DOI: 10.1289/EHP1275.].

BACKGROUND: Formaldehyde (FA) is an environmental and occupational chemical carcinogen. Recent studies have shown that exogenous FA causes only a modest increase in DNA adduct formation compared with the amount of adducts formed by endogenous FA, raising the possibility that epigenetic mechanisms may contribute to FA-mediated carcinogenicity. OBJECTIVES: We investigated the effects of FA exposure on histone modifications and chromatin assembly. We also examined the role of defective chromatin assembly in FA-mediated transcription and cell transformation. METHODS: Cellular fractionation and Western blot analysis were used to measure the levels of histone modifications in human bronchial epithelial BEAS-2B cells and human nasal RPMI2650 cells in the presence of FA. Chromatin immunoprecipitation (ChIP) and micrococcal nuclease (MNase) digest assays were performed to examine the changes in chromatin assembly and accessibility after FA exposure. RNA sequencing (RNA-seq) and real-time polymerase chain reaction (PCR) were used to examine transcriptional dysregulation. Finally, anchorage-independent cell growth ability was tested by soft agar assay following FA exposure. RESULTS: Exposure to FA dramatically decreased the acetylation of the N-terminal tails of cytosolic histones. These modifications are important for histone nuclear import and subsequent chromatin assembly. Histone proteins were depleted in both the chromatin fraction and at most of the genomic loci tested following FA exposure, suggesting that FA compromises chromatin assembly. Moreover, FA increased chromatin accessibility and altered the expression of hundreds of cancer-related genes. Knockdown of the histone H3.3 gene (an H3 variant), which mimics inhibition of chromatin assembly, facilitated FA-mediated anchorage-independent cell growth. CONCLUSIONS: We propose that the inhibition of chromatin assembly represents a novel mechanism of cell transformation induced by the environmental and occupational chemical carcinogen FA. https://doi.org/10.1289/EHP1275.