Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - CRISTONE (Targeting CRISPR-based HDAC inhibitors to histones: a new tool for assessing mechanisms of class I HDAC inhibitors and developing chemical probes.)

Teaser

Epigenetic regulation comprises numerous mechanisms providing regulatory information to the genome without altering its primary nucleotide sequence, information transferred heritably to offspring or from parent to daughter cells. Epigenetic mechanisms are implicated in various...

Summary

Epigenetic regulation comprises numerous mechanisms providing regulatory information to the genome without altering its primary nucleotide sequence, information transferred heritably to offspring or from parent to daughter cells. Epigenetic mechanisms are implicated in various processes, including gene silencing and expression, apoptosis, maintenance of stem cell pluripotency, and X-chromosome inactivation. At the molecular level, epigenetic regulators include covalent modifications to the chromatin, which is composed of DNA in complex with proteins, mainly histones. This family of small proteins is usually represented by histones H1, H2A, H2B, H3 and H4, which in turn are composed of basic amino acids necessary for DNA binding. Post-translational modifications of histones (e.g., histone acetylation, methylation, and ubiquitination) or DNA (e.g., DNA methylation) locally modify the chromatin structure by altering the interaction between histones and DNA. The altered states can either facilitate or prevent gene transcription. Histone acetylation leads to chromatin relaxation and increased accessibility for transcription factors. On the contrary, deacetylation by histone deacetylases (HDACs) has a repressive impact on transcription. Among HDAC enzymes, class I comprises the constitutively expressed HDAC1-3 and HDAC8, having histones as their main substrates.
Given their nuclear localization, class I HDACs are among the key regulators responsible for epigenetic marks. Because several studies suggest that HDAC overexpression is linked to various diseases (such as cancer, cardiovascular, and neurodegenerative disorders), HDAC inhibition has emerged as an attractive therapeutic strategy to restore the histone acetylation balance. However, it is difficult to target drugs influencing histone-modifying enzymes to specific genomic loci. Moreover, HDAC inhibitors are known to have effects on the acetylation of other proteins. To have a direct effect on histone acetylation leading to transcriptional activation, HDAC inhibitors need to cross multiple cell membranes to reach the nucleus, avoid interaction with other lysine deacetylating enzymes, and avoid binding to HDAC isoforms located in the cytoplasm. Furthermore, the cellular effects of deacetylation inhibition on non-histone proteins need to be discriminated when trying to understand the effects of the HDAC inhibitors.
HDAC inhibitors are currently used in cancer therapy and have been considered as epigenetic drugs. However, whether the therapeutic effects of HDAC inhibitors are a direct consequence of changes in chromatin accessibility and transcription remains to be proven. Together with the drug localization issue, the specific mechanisms involved in therapeutic success/failure of HDAC inhibitors remain to be investigated. These mechanisms include i) the genome-wide transcriptional effects resulting from histone deacetylation in cells overexpressing specific HDAC isoforms; ii) the effects of isoform-dependent transcriptional regulation and their association with chromatin accessibility; and iii) the effects of HDAC inhibitory drugs that are a direct consequence of loci-related chromatin remodeling. This project aimed to address the issues mentioned above through an innovative approach, using HDAC-overexpressing cells, genome-wide studies, and a new epigenetic editing tool (CRISTONE) designed to bring an HDAC inhibitor to a specific locus using the guide-RNA-targeted CRISPR/dCas9 technology. This will be useful in the search for new cancer treatment, including epigenetic therapies.

Work performed

The first step achieved was the generation of cell lines for the inducible overexpression of the class I histone deacetylases HDAC1, HDAC3, and HDAC8. The histone acetylation profile was evaluated for each cell line, before and after induction of overexpression, employing Western blot and mass spectrometry analyses. Accordingly, the genome-wide expression profile of each cell line was analyzed by RNA-seq and their chromatin accessibility by ATAC-seq. A locus linked to the promoter of CDKN1A gene was selected in the HDAC1 overexpressing cells to be targeted by CRISTONE. The cloning steps of CRISTONE were established and CRISTONE vectors were obtained. A guide-RNA vector was designed to target the promoter of CDKN1A. The genome-wide transcriptional effects of the distinct components of the CRISPR/dCas9 system were investigated by RNA-seq and confirmed by qRT-PCR. All generated data and vectors will be made available by repository tools after publication in open access journals. The researcher has already submitted 3 articles for publication (currently under review), one as the main author, and the others as a co-author. An additional scientific manuscript is currently being prepared, contributing to the visibility of the researcher, the host institutions and the European Commission. The results being considered for publication are of great contribution to the field of technological applications and pitfalls of CRISPR epigenetic editing. These dissemination tasks are pertinent at a time when CRISPR technologies are being applied to commercial products (e.g. reagents, genetically modified organisms) and for therapeutic purposes in clinical trials.

Final results

Several differences have been noticed in the histone acetylation profile upon activation of specific HDAC isoforms, showing that distinct HDAC isoforms have different endogenous histone tail peptides as substrates. The sequencing results (RNA-seq and ATAC-seq) also showed specific effects of gene expression relative to each HDAC isoform. These findings corroborate the idea that isoform-specific inhibitors may be valuable as chemical probes and, eventually, in drug therapy. The gene selected in the HDAC1 overexpressing cells (CDKN1A) has been previously demonstrated to be controlled by HDACs and chemical HDAC inhibitors. Therefore, it is an ideal probe for testing the effect of the CRISTONE tool aiming at answering the questions on the real mechanisms of HDAC inhibitors, a step to be pursued using the CRISPR-based CRISTONE tool. However, previous studies have suggested that CRISPR components induce cellular side effects. When used for epigenetic editing, CRISPR side effects may confound outcomes, especially if they are maintained through cell divisions. Therefore, it was essential to investigate the side effects of the epigenetic editing tool in the human parental cell line. Such investigation showed that CRISPR components triggered transcriptional dysregulation, resulting in the induction of stress-response genes uncoupled from the guide-RNA targeted gene. These results are relevant to the scientific community working with CRISPR editing tools at a time when CRISPR technologies are being applied to commercial products (e.g., reagents, genetically modified organisms) and for therapeutic purposes in clinical trials, with an impact on societal, ethical, and safety issues.