Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.

Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.

Mouse Heterochromatin Adopts Digital Compaction States without Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separation.

The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.

Epigenetic inheritance is gated by naïve pluripotency and Dppa2.

Environmental factors can trigger cellular responses that propagate across mitosis or even generations. Perturbations to the epigenome could underpin such acquired changes, however, the extent and contexts in which modified chromatin states confer heritable memory in mammals is unclear. Here, we exploit a precision epigenetic editing strategy and forced Xist activity to programme de novo heterochromatin domains (epialleles) at endogenous loci and track their inheritance in a developmental model. We find that naïve pluripotent phases systematically erase ectopic domains of heterochromatin via active mechanisms, which likely acts as an intergenerational safeguard against transmission of epialleles. Upon lineage specification, however, acquired chromatin states can be probabilistically inherited under selectively favourable conditions, including propagation of p53 silencing through in vivo development. Using genome-wide CRISPR screening, we identify molecular factors that restrict heritable memory of epialleles in naïve pluripotent cells, and demonstrate that removal of chromatin factor Dppa2 unlocks the potential for epigenetic inheritance uncoupled from DNA sequence. Our study outlines a mechanistic basis for how epigenetic inheritance is constrained in mammals, and reveals genomic and developmental contexts in which heritable memory is feasible.

CRISPR/dCas9 DNA methylation editing is heritable during human hematopoiesis and shapes immune progeny.

Aging is associated with an abnormal increase in DNA methylation (DNAm) in human gene promoters, including in bone marrow stem cells. DNAm patterns are further perturbed in hematological malignancies such as acute myeloid leukemia but the physiological significance of such epigenetic changes is unknown. Using epigenetic editing of human stem/progenitor cells (HSPCs), we show that p15 methylation affects hematopoiesis in vivo. We edited the CDKN2B (p15) promoter and ARF (p14) using dCas9-3A3L and observed DNAm spreading beyond the gRNA location. We find that despite a transient delivery system, DNAm is maintained during myeloid differentiation in vitro, and hypermethylation of the p15 promoter reduces gene expression. In vivo, edited human HSPCs can engraft the bone marrow of mice and targeted DNAm is maintained in HSPCs long term. Moreover, epigenetic changes are conserved and inherited in both myeloid and lymphoid lineages. Although the proportion of myeloid (CD33+) and lymphoid (CD19+) cells is unaffected, monocyte (CD14+) populations decreased and granulocytes (CD66b+) increased in mice engrafted with p15 hypermethylated HSPCs. Monocytes derived from p15 hypermethylated HSPCs appear to be activated and show increased inflammatory transcriptional programs. We believe these findings have clinical relevance since we found p15 promoter methylation in the peripheral blood of patients with clonal hematopoiesis. Our study shows DNAm can be targeted and maintained in human HSPCs and demonstrated functional relevance of aberrant DNAm on the p15 locus. As such, other aging-associated aberrant DNAm may impact hematopoiesis in vivo.

Targeting epigenetic aberrations of sarcoma in CRISPR era.

Sarcomas are rare malignancies that exhibit diverse biological, genetic, morphological, and clinical characteristics. Genetic alterations, such as gene fusions, mutations in transcriptional machinery components, histones, and DNA methylation regulatory molecules, play an essential role in sarcomagenesis. These mutations induce and/or cooperate with specific epigenetic aberrations required for the growth and maintenance of sarcomas. Appropriate mouse models have been developed to clarify the significance of genetic and epigenetic interactions in sarcomas. Studies using the mouse models for human sarcomas have demonstrated major advances in our understanding the developmental processes as well as tumor microenvironment of sarcomas. Recent technological progresses in epigenome editing will not only improve the studies using animal models but also provide a direct clue for epigenetic therapies. In this manuscript, we review important epigenetic aberrations in sarcomas and their representative mouse models, current methods of epigenetic editing using CRISPR/dCas9 systems, and potential applications in sarcoma studies and therapeutics.

Unveiling the epigenomic mechanisms of acquired platinum-resistance in high-grade serous ovarian cancer.

Resistance to platinum-based chemotherapy is the major cause of death from high-grade serous ovarian cancer (HGSOC). We hypothesise that detection of specific DNA methylation changes may predict platinum resistance in HGSOC. Using a publicly available "discovery" dataset we examined epigenomic and transcriptomic alterations between primary platinum-sensitive (n = 32) and recurrent acquired drug resistant HGSOC (n = 28) and identified several genes involved in immune and chemoresistance-related pathways. Validation via high-resolution melt analysis of these findings, in cell lines and HGSOC tumours, demonstrated the most consistent changes were observed in three of the genes: APOBEC3A, NKAPL and PDCD1. Plasma samples from an independent HGSOC cohort (n = 17) were analysed using droplet digital PCR. Hypermethylation of NKAPL was detected in 46% and hypomethylation of APOBEC3A in 69% of plasma samples taken from women with relapsed HGSOC (n = 13), with no alterations identified in disease-free patients (n = 4). Following these results, and using a CRISPR-Cas9 approach, we were also able to demonstrate that in vitro NKAPL promoter demethylation increased platinum sensitivity by 15%. Overall, this study demonstrates the importance of aberrant methylation, especially of the NKAPL gene, in acquired platinum resistance in HGSOC.

From methylation to myelination: epigenomic and transcriptomic profiling of chronic inactive demyelinated multiple sclerosis lesions.

In the progressive phase of multiple sclerosis (MS), the hampered differentiation capacity of oligodendrocyte precursor cells (OPCs) eventually results in remyelination failure. We have previously shown that DNA methylation of Id2/Id4 is highly involved in OPC differentiation and remyelination. In this study, we took an unbiased approach by determining genome-wide DNA methylation patterns within chronically demyelinated MS lesions and investigated how certain epigenetic signatures relate to OPC differentiation capacity. We compared genome-wide DNA methylation and transcriptional profiles between chronically demyelinated MS lesions and matched normal-appearing white matter (NAWM), making use of post-mortem brain tissue (n = 9/group). DNA methylation differences that inversely correlated with mRNA expression of their corresponding genes were validated for their cell-type specificity in laser-captured OPCs using pyrosequencing. The CRISPR-dCas9-DNMT3a/TET1 system was used to epigenetically edit human-iPSC-derived oligodendrocytes to assess the effect on cellular differentiation. Our data show hypermethylation of CpGs within genes that cluster in gene ontologies related to myelination and axon ensheathment. Cell type-specific validation indicates a region-dependent hypermethylation of MBP, encoding for myelin basic protein, in OPCs obtained from white matter lesions compared to NAWM-derived OPCs. By altering the DNA methylation state of specific CpGs within the promotor region of MBP, using epigenetic editing, we show that cellular differentiation and myelination can be bidirectionally manipulated using the CRISPR-dCas9-DNMT3a/TET1 system in vitro. Our data indicate that OPCs within chronically demyelinated MS lesions acquire an inhibitory phenotype, which translates into hypermethylation of crucial myelination-related genes. Altering the epigenetic status of MBP can restore the differentiation capacity of OPCs and possibly boost (re)myelination.

Tailored chromatin modulation to promote tissue regeneration.

Epigenetic regulation of gene expression is fundamental in the maintenance of cellular identity and the regulation of cellular plasticity during tissue repair. In fact, epigenetic modulation is associated with the processes of cellular de-differentiation, proliferation, and re-differentiation that takes place during tissue regeneration. In here we explore the epigenetic events that coordinate tissue repair in lower vertebrates with high regenerative capacity, and in mammalian adult stem cells, which are responsible for the homeostasis maintenance of most of our tissues. Finally we summarize promising CRISPR-based editing technologies developed during the last years, which look as promising tools to not only study but also promote specific events during tissue regeneration.

Targeting cancer epigenetics with CRISPR-dCAS9: Principles and prospects.

Cancer therapeutics is an ever-evolving field due to incessant demands for effective and precise treatment options. Over the last few decades, cancer treatment strategies have shifted somewhat from surgery to targeted precision medicine. CRISPR-dCas9 is an emerging version of precision cancer therapy that has been adapted from the prokaryotic CRISPR-Cas system. Once ligated to epigenetic effectors (EE), CRISPR-dCas9 can function as an epigenetic editing tool and CRISPR-dCas9-EE complexes could be exploited to alter cancerous epigenetic features associated with different cancer hallmarks. In this article, we discuss the rationale of epigenetic editing as a therapeutic strategy against cancer. We also outline how sgRNA-dCas9 was derived from the CRISPR-Cas system. In addition, the current status of sgRNA-dCas9 use (in vivo and in vitro) in cancer is updated with a molecular illustration of CRISPR-dCas9-mediated epigenetic and transcriptional modulation. As sgRNA-dCas9 is still at the developmental phase, challenges are inherent to its use. We evaluate major challenges in targeting cancer with sgRNA-dCas9 such as off-target effects, lack of sgRNA designing rubrics, target site selection dilemmas and deficient sgRNA-dCas9 delivery systems. Finally, we appraise the sgRNA-dCas9 as a prospective cancer therapeutic by summarizing ongoing improvements of sgRNA-dCas9 methodology.

Gene-Targeted DNA Methylation: Towards Long-Lasting Reprogramming of Gene Expression?

DNA methylation is an essential epigenetic mark, strongly associated with gene expression regulation. Aberrant DNA methylation patterns underlie various diseases and efforts to intervene with DNA methylation signatures are of great clinical interest. Technological developments to target writers or erasers of DNA methylation to specific genomic loci by epigenetic editing resulted in successful gene expression modulation, also in in vivo models. Application of epigenetic editing in human health could have a huge impact, but clinical translation is still challenging. Despite successes for a wide variety of genes, not all genes mitotically maintain their (de)methylation signatures after editing, and reprogramming requires further understanding of chromatin context-dependency. In addition, difficulties of current delivery systems and off-target effects are hurdles to be tackled. The present review describes findings towards effective and sustained DNA (de)methylation by epigenetic editing and discusses the need for multi-effector approaches to achieve highly efficient long-lasting reprogramming.

Epigenetic editing by CRISPR/dCas9 in Plasmodium falciparum.

Genetic manipulation remains a major obstacle for understanding the functional genomics of the deadliest malaria parasite Plasmodium falciparum Although the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system has been successfully applied to introduce permanent changes in the parasite genome, its use is still limited. Here we show that fusing different epigenetic effector domains to a Cas9 null mutant efficiently and specifically reprograms the expression of target genes in P. falciparum By precisely writing and erasing histone acetylation at the transcription start site regions of the invasion-related genes reticulocyte binding protein homolog 4 (rh4) and erythrocyte binding protein 175 (eba-175), respectively, we achieved significant activation of rh4 and repression of eba-175, leading to the switch of the parasite invasion pathways into human erythrocytes. By using the epigenetic knockdown system, we have also characterized the effects of PfSET1, previously identified as an essential gene, on expression of mainly trophozoite- and schizont-specific genes, and therefore regulation of the growth of the mature forms of P. falciparum This epigenetic CRISPR/dCas9 system provides a powerful approach for regulating gene expression at the transcriptional level in P. falciparum.

Advances of Epigenetic Biomarkers and Epigenome Editing for Early Diagnosis in Breast Cancer.

Epigenetic modifications are known to regulate cell phenotype during cancer progression, including breast cancer. Unlike genetic alterations, changes in the epigenome are reversible, thus potentially reversed by epi-drugs. Breast cancer, the most common cause of cancer death worldwide in women, encompasses multiple histopathological and molecular subtypes. Several lines of evidence demonstrated distortion of the epigenetic landscape in breast cancer. Interestingly, mammary cells isolated from breast cancer patients and cultured ex vivo maintained the tumorigenic phenotype and exhibited aberrant epigenetic modifications. Recent studies indicated that the therapeutic efficiency for breast cancer regimens has increased over time, resulting in reduced mortality. Future medical treatment for breast cancer patients, however, will likely depend upon a better understanding of epigenetic modifications. The present review aims to outline different epigenetic mechanisms including DNA methylation, histone modifications, and ncRNAs with their impact on breast cancer, as well as to discuss studies highlighting the central role of epigenetic mechanisms in breast cancer pathogenesis. We propose new research areas that may facilitate locus-specific epigenome editing as breast cancer therapeutics.

Epigenetic Mechanisms in Parenchymal Lung Diseases: Bystanders or Therapeutic Targets?

Epigenetic responses due to environmental changes alter chromatin structure, which in turn modifies the phenotype, gene expression profile, and activity of each cell type that has a role in the pathophysiology of a disease. Pulmonary diseases are one of the major causes of death in the world, including lung cancer, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), pulmonary hypertension (PH), lung tuberculosis, pulmonary embolism, and asthma. Several lines of evidence indicate that epigenetic modifications may be one of the main factors to explain the increasing incidence and prevalence of lung diseases including IPF and COPD. Interestingly, isolated fibroblasts and smooth muscle cells from patients with pulmonary diseases such as IPF and PH that were cultured ex vivo maintained the disease phenotype. The cells often show a hyper-proliferative, apoptosis-resistant phenotype with increased expression of extracellular matrix (ECM) and activated focal adhesions suggesting the presence of an epigenetically imprinted phenotype. Moreover, many abnormalities observed in molecular processes in IPF patients are shown to be epigenetically regulated, such as innate immunity, cellular senescence, and apoptotic cell death. DNA methylation, histone modification, and microRNA regulation constitute the most common epigenetic modification mechanisms.

Epigenetic Changes and Chromatin Reorganization in Brain Function: Lessons from Fear Memory Ensemble and Alzheimer's Disease.

Healthy brain functioning in mammals requires a continuous fine-tuning of gene expression. Accumulating evidence over the last three decades demonstrates that epigenetic mechanisms and dynamic changes in chromatin organization are critical components during the control of gene transcription in neural cells. Recent genome-wide analyses show that the regulation of brain genes requires the contribution of both promoter and long-distance enhancer elements, which must functionally interact with upregulated gene expression in response to physiological cues. Hence, a deep comprehension of the mechanisms mediating these enhancer-promoter interactions (EPIs) is critical if we are to understand the processes associated with learning, memory and recall. Moreover, the onset and progression of several neurodegenerative diseases and neurological alterations are found to be strongly associated with changes in the components that support and/or modulate the dynamics of these EPIs. Here, we overview relevant discoveries in the field supporting the role of the chromatin organization and of specific epigenetic mechanisms during the control of gene transcription in neural cells from healthy mice subjected to the fear conditioning paradigm, a relevant model to study memory ensemble. Additionally, special consideration is dedicated to revising recent results generated by investigators working with animal models and human postmortem brain tissue to address how changes in the epigenome and chromatin architecture contribute to transcriptional dysregulation in Alzheimer's disease, a widely studied neurodegenerative disease. We also discuss recent developments of potential new therapeutic strategies involving epigenetic editing and small chromatin-modifying molecules (or epidrugs).

Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells.

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available. In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (ß-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells. Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level.

Correction of Heritable Epigenetic Defects Using Editing Tools.

Epimutations refer to mistakes in the setting or maintenance of epigenetic marks in the chromatin. They lead to mis-expression of genes and are often secondary to germline transmitted mutations. As such, they are the cause for a considerable number of genetically inherited conditions in humans. The correction of these types of epigenetic defects constitutes a good paradigm to probe the fundamental mechanisms underlying the development of these diseases, and the molecular basis for the establishment, maintenance and regulation of epigenetic modifications in general. Here, we review the data to date, which is limited to repetitive elements, that relates to the applications of key editing tools for addressing the epigenetic aspects of various epigenetically regulated diseases. For each approach we summarize the efforts conducted to date, highlight their contribution to a better understanding of the molecular basis of epigenetic mechanisms, describe the limitations of each approach and suggest perspectives for further exploration in this field.

Silencing integrated SIV proviral DNA with TAR-specific CRISPR tools.

BACKGROUND: One approach for a functional HIV cure is to prevent transcription from integrated proviral DNA. A critical step in HIV transcription is the Tat protein interaction with the TAR element viral RNA. We tested the strategy of blocking this Tat-TAR interaction in the SIVmac model. METHODS: We designed five CRISPR short guiding RNAs (sgRNAs) targeting the SIVmac TAR element, along with inactive versions of Cas9 (dCas9). These sgRNA constructs were delivered as ribonucleoproteins or plasmid DNA, along with SIV DNA. The constructs were also tested in integrated viral DNA in a cell line chronically infected by SIV. RESULTS: The sgRNAs targeting the coding strand of the TAR element inhibited SIV RNA transcription in association with dCas9-KRAB, but not with dCas9. CONCLUSIONS: Induction of epigenetic modifications may be more effective in inactivating provirus than transcriptional interference and thus may be a better strategy to achieve a functional cure in vivo.

KRAB-Induced Heterochromatin Effectively Silences PLOD2 Gene Expression in Somatic Cells and is Resilient to TGFß1 Activation.

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFß1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFß1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFß1-induced and uninduced PLOD2 expression.

Targeting Epigenetic Modifications in Uveal Melanoma.

Uveal melanoma (UM), the most common intraocular malignancy in adults, is a rare subset of melanoma. Despite effective primary therapy, around 50% of patients will develop the metastatic disease. Several clinical trials have been evaluated for patients with advanced UM, though outcomes remain dismal due to the lack of efficient therapies. Epigenetic dysregulation consisting of aberrant DNA methylation, histone modifications, and small non-coding RNA expression, silencing tumor suppressor genes, or activating oncogenes, have been shown to play a significant role in UM initiation and progression. Given that there is no evidence any approach improves results so far, adopting combination therapies, incorporating a new generation of epigenetic drugs targeting these alterations, may pave the way for novel promising therapeutic options. Furthermore, the fusion of effector enzymes with nuclease-deficient Cas9 (dCas9) in clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) system equips a potent tool for locus-specific erasure or establishment of DNA methylation as well as histone modifications and, therefore, transcriptional regulation of specific genes. Both, CRISPR-dCas9 potential for driver epigenetic alterations discovery, and possibilities for their targeting in UM are highlighted in this review.

A translational perspective on epigenetics in allergic diseases.

The analysis of epigenetic modifications in allergic diseases has recently attracted substantial interest because epigenetic modifications can mediate the effects of the environment on the development of or protection from allergic diseases. Furthermore, recent research has provided evidence for an altered epigenomic landscape in disease-relevant cell populations. Although still in the early phase, epigenetic modifications, particularly DNA methylation and microRNAs, might have potential for assisting in the stratification of patients for treatment and complement or replace in the future biochemical or clinical tests. The first epigenetic biomarkers correlating with the successful outcome of immunotherapy have been reported, and with personalized treatment options being rolled out, epigenetic modifications might well play a role in monitoring or even predicting the response to tailored therapy. However, further studies in larger cohorts with well-defined phenotypes in specific cell populations need to be performed before their implementation. Furthermore, the epigenome provides an interesting target for therapeutic intervention, with microRNA mimics, inhibitors, and antisense oligonucleotides being evaluated in clinical trials in patients with other diseases. Selection or engineering of populations of extracellular vesicles and epigenetic editing represent novel tools for modulation of the cellular phenotype and responses, although further technological improvements are required. Moreover, interactions between the host epigenome and the microbiome are increasingly recognized, and interventions of the microbiome could contribute to modulation of the epigenome with a potential effect on the overall goal of prevention of allergic diseases.