Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors. This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment. The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting. The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.
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http://dx.doi.org/10.3791/61289 | DOI Listing |
Cell Signal
January 2025
Institute of Medical Science, Ajou University School of Medicine, Suwon, Gyeonggi 16499, Republic of Korea; Department of Biomedical Sciences, Ajou University Graduate School of Medicine, Suwon, Gyeonggi 16499, Republic of Korea. Electronic address:
Oxidative stress caused by reactive oxygen species (ROS) and superoxides is linked to various cancer-related biological events. Extracellular superoxide dismutase (SOD3), an antioxidant enzyme that removes superoxides, contributes to redox homeostasis and has the potential to regulate tumorigenesis. Histone deacetylase 6 (HDAC6), a major HDAC isoform responsible for mediating the deacetylation of non-histone protein substrates, also plays a role in cancer progression.
View Article and Find Full Text PDFNeurochem Res
January 2025
Huazhong University of Science and Technology, Tongji Medical College, Wuhan, Hubei, 430000, China.
Epilepsy (EP) is a neurological disorder characterized by abnormal, sudden neuronal discharges. Seizures increase extracellular glutamate levels, causing excitotoxic damage. Glutamate transporter type 1 (GLT-1) and its human homologue excitatory amino acid transporter-2 (EAAT2) clear 95% of extracellular glutamate.
View Article and Find Full Text PDFClin Epigenetics
January 2025
Department of Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
Background: The primary objective of this study was to examine whether ARID1A mutations confer a fitness advantage to gastric cancer from an immunological perspective, along with elucidating the underlying mechanism. Additionally, we aimed to identify the clinical potential of combining epigenetic inhibitors with immune checkpoint inhibitors to improve the efficacy of immunotherapy for gastric cancer.
Methods: The correlation between ARID1A gene expression and gastric cancer patient survival was analyzed using the GEO dataset GSE62254.
Alzheimers Dement
December 2024
Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
Background: Aging is a time-dependent deterioration of physiological functions that occurs in both humans and animals. Within the brain, aging cells gradually become dysfunctional through a complex interplay of intrinsic and extrinsic factors, ultimately leading to behavioral deficits and enhanced risk of neurodegenerative diseases such as Alzheimer's disease (AD). The characteristics of normal aging are distinct from those associated with age-related diseases and it is important to understand the processes that contribute to this pathological divergence.
View Article and Find Full Text PDFBackground: In Alzheimer's disease (AD), histone acetylation is disrupted, suggesting loss of transcriptional control. Moreover, converging evidence suggests an age- and AD-dependent loss of transcription controlled by all-trans-retinoic acid (ATRA), the bioactive metabolite of vitamin A (VA). Antioxidant depletion causes oxidative stress (OS).
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