Histone H4 asymmetrically dimethylated at arginine 3 (H4R3me2a), a mark of super-enhancers.

Histone H4 asymmetrically dimethylated at arginine 3 (H4R3me2a) is an active histone mark catalyzed by protein arginine methyltransferase 1 (PRMT1), a major arginine methyltransferase in vertebrates catalyzing asymmetric dimethylation of arginine. H4R3me2a stimulates the activity of lysine acetyltransferases such as CBP/p300, which catalyze the acetylation of H3K27, a mark of active enhancers, super-enhancers, and promoters. There are a few studies on the genomic location of H4R3me2a. In chicken polychromatic erythrocytes, H4R3me2a is found in introns and intergenic regions and binds to the globin locus control region (a super-enhancer) and globin regulatory regions. In this report, we analyzed chromatin immunoprecipitation sequencing data for the genomic location of H4R3me2a in the breast cancer cell line MCF7. As in avian cells, MCF7 H4R3me2a is present in intronic and intergenic regions. Nucleosomes with H4R3me2a and H3K27ac next to nucleosome-free regions are found at super-enhancers, enhancers, and promoter regions of expressed genes. Genes with critical roles in breast cancer cells have broad domains of nucleosomes with H4R3me2a, H3K27ac, and H3K4me3. Our results are consistent with PRMT1-mediated H4R3me2a playing a key role in the chromatin organization of regulatory regions of vertebrate genomes.

Broad histone H4 monomethylation marks expressed genes involved in translation.

H4K20me1 (histone H4 monomethylated at lysine 20) generally has a broad distribution along genes and has been reported to be associated with expressed and repressed genes. In contrast, H3K4me3 (histone H3 trimethylated at lysine 4) is positioned as a narrow peak at the 5' end of most expressed genes in vertebrate cells. A small population of genes involved in cell identity has H3K4me3 distributed throughout the gene body. In this report, we show that H4K20me1 is associated with expressed genes in estrogen receptor-positive breast cancer MCF7 cells and erythroleukemic K562 cells. Further, we identified the genes with the broadest H4K20me1 domains in these two cell types. The broad H4K20me1 domain marked gene bodies of expressed genes, but not the promoter or enhancer regions. The most significant GO term (biological processes) of these genes was cytoplasmic translation. There was little overlap between the genes marked with the broad H4K20me1 domain and those marked with H3K4me3. H4K20me1 and H3K79me2 distributions along expressed gene bodies were similar, suggesting a relationship between the enzymes catalyzing these histone modifications.

Mitogen- and stress-activated protein kinase (MSK1/2) regulated gene expression in normal and disease states.

The mitogen- and stress-activated protein kinases (MSK) are epigenetic modifiers that regulate gene expression in normal and disease cell states. MSK1 and 2 are involved in a chain of signal transduction events bringing signals from the external environment of a cell to specific sites in the genome. MSK1/2 phosphorylate histone H3 at multiple sites, resulting in chromatin remodeling at regulatory elements of target genes and the induction of gene expression. Several transcription factors (RELA of NF-κB and CREB) are also phosphorylated by MSK1/2 and contribute to induction of gene expression. In response to signal transduction pathways, MSK1/2 can stimulate genes involved in cell proliferation, inflammation, innate immunity, neuronal function, and neoplastic transformation. Abrogation of the MSK-involved signaling pathway is among the mechanisms by which pathogenic bacteria subdue the host's innate immunity. Depending on the signal transduction pathways in play and the MSK-targeted genes, MSK may promote or hinder metastasis. Thus, depending on the type of cancer and genes involved, MSK overexpression may be a good or poor prognostic factor. In this review, we focus on mechanisms by which MSK1/2 regulate gene expression, and recent studies on their roles in normal and diseased cells.

Integrative analysis reveals novel associations between DNA methylation and the serum metabolome of adolescents with type 2 diabetes: A cross-sectional study.

Objective: Rates of type 2 diabetes (T2D) among adolescents are on the rise. Epigenetic changes could be associated with the metabolic alterations in adolescents with T2D. Methods: We performed a cross sectional integrated analysis of DNA methylation data from peripheral blood mononuclear cells with serum metabolomic data from First Nation adolescents with T2D and controls participating in the Improving Renal Complications in Adolescents with type 2 diabetes through Research (iCARE) cohort study, to explore the molecular changes in adolescents with T2D. Results: Our analysis showed that 43 serum metabolites and 36 differentially methylated regions (DMR) were associated with T2D. Several DMRs were located near the transcriptional start site of genes with established roles in metabolic disease and associated with altered serum metabolites (e.g. glucose, leucine, and gamma-glutamylisoleucine). These included the free fatty acid receptor-1 (FFAR1), upstream transcription factor-2 (USF2), and tumor necrosis factor-related protein-9 (C1QTNF9), among others. Conclusions: We identified DMRs and metabolites that merit further investigation to determine their significance in controlling gene expression and metabolism which could define T2D risk in adolescents.

Bioinformatic Analyses of Broad H3K79me2 Domains in Different Leukemia Cell Line Data Sets.

A subset of expressed genes is associated with a broad H3K4me3 (histone H3 trimethylated at lysine 4) domain that extends throughout the gene body. Genes marked in this way in normal cells are involved in cell-identity and tumor-suppressor activities, whereas in cancer cells, genes driving the cancer phenotype (oncogenes) have this feature. Other histone modifications associated with expressed genes that display a broad domain have been less studied. Here, we identified genes with the broadest H3K79me2 (histone H3 dimethylated at lysine 79) domain in human leukemic cell lines representing different forms of leukemia. Taking a bioinformatic approach, we provide evidence that genes with the broadest H3K79me2 domain have known roles in leukemia (e.g., JMJD1C). In the mixed-lineage leukemia cell line MOLM-13, the HOXA9 gene is in a 100 kb broad H3K79me2 domain with other HOXA protein-coding and oncogenic long non-coding RNA genes. The genes in this domain contribute to leukemia. This broad H3K79me2 domain has an unstable chromatin structure, as was evident by enhanced chromatin accessibility throughout. Together, we provide evidence that identification of genes with the broadest H3K79me2 domain will aid in generating a panel of genes in the diagnosis and therapeutic treatment of leukemia in the future.

The key role of differential broad H3K4me3 and H3K4ac domains in breast cancer.

Epigenetic processes are radically altered in cancer cells. The altered epigenetic events may include histone post-translational modifications (PTMs), DNA modifications, and/or alterations in the levels and modifications of chromatin modifying enzymes and chromatin remodelers. With changes in gene programming are changes in the genomic distribution of histone PTMs. Genes that are poised or transcriptionally active have histone H3 trimethylated lysine 4 (H3K4me3) located at the transcription start site and at the 5' end of the gene. However, a small population of genes that are involved in cell identity or cancer cell properties have a broad H3K4me3 domain that may stretch for several kilobases through the coding region of the gene. Each cancer cell type appears to mark a select set of cancer-related genes in this manner. In this study, we determined which genes were differentially marked with the broad H3K4me3 domain in normal-like (MCF10A), luminal-type breast cancer (MCF7), and triple-negative breast cancer (MDA-MB-231) cells. We also determined whether histone H3 acetylated lysine 4 (H3K4ac), also a mark of active promoters, had a broad domain configuration. We applied two peak callers (MACS2, PeakRanger) to analyze H3K4me3 and H3K4ac chromatin immunoprecipitation sequencing (ChIP-Seq) data. We identified genes with a broad H3K4me3 and/or H3K4ac domain specific to each cell line and show that the genes have critical roles in the breast cancer subtypes. Furthermore, we show that H3K4ac marks enhancers. The identified genes with the broad H3K4me3/H3K4ac domain have been targeted in clinical and pre-clinical studies including therapeutic treatments of breast cancer.

Differential expression of HNF1A and HNF1A-AS1 in colon cancer cells.

The human hepatocyte nuclear factor 1 homeobox A (HNF1A) gene loci express the protein-coding HNF1A transcript and a long non-coding RNA in the anti-sense (HNF1A-AS1) direction. HNF1A-AS1 is expressed in numerous types of cancers and poor clinical outcomes such as higher mortality rates, greater metastatic capacity, and poor prognosis of the disease are the results of this expression. In this study, we determined the epigenetic features of the HNF1A gene loci, and expression and cellular localization of HNF1A-AS1 RNA, HNF1A RNA, and HNF1A protein in colorectal cancer (HT-29, HTC116, RKO, and SW480) and normal colon epithelial (CCD841) cells. The HT-29 HNF1A gene had active histone marks (H3K4me3, H3K27ac) and DNase 1 accessible sites at the promoter regions of the HNF1A and HNF1A-AS1 genes. These epigenetic marks were not observed in the other colorectal cancer cells or in the normal colon epithelial cells. Consistent with the active gene epigenetic signature of the HNF1A gene in HT-29 cells, HNF1A protein, and HNF1A/HNF1A-AS1 transcripts were detected in HT-29 cells but poorly, if at all observed, in the other cell types. In HT-29 cells, HNF1A-AS1 localized to the nucleus and was found to bind to the enhancer of zeste homolog 2 (EZH2, a member of PRC2 complex) and potentially form RNA-DNA triplexes with DNase 1 accessible sites in the HT-29 genome. These activities of HNF1A-AS1 may contribute to the oncogenic properties of this long non-coding RNA.

The dynamic broad epigenetic (H3K4me3, H3K27ac) domain as a mark of essential genes.

Transcriptionally active chromatin is marked by tri-methylation of histone H3 at lysine 4 (H3K4me3) located after first exons and around transcription start sites. This epigenetic mark is typically restricted to narrow regions at the 5`end of the gene body, though a small subset of genes have a broad H3K4me3 domain which extensively covers the coding region. Although most studies focus on the H3K4me3 mark, the broad H3K4me3 domain is associated with a plethora of histone modifications (e.g., H3 acetylated at K27) and is therein termed broad epigenetic domain. Genes marked with the broad epigenetic domain are involved in cell identity and essential cell functions and have clinical potential as biomarkers for patient stratification. Reducing expression of genes with the broad epigenetic domain may increase the metastatic potential of cancer cells. Enhancers and super-enhancers interact with the broad epigenetic domain marked genes forming a hub of interactions involving nucleosome-depleted regions. Together, the regulatory elements coalesce with transcription factors, chromatin modifying/remodeling enzymes, coactivators, and the Mediator and/or Integrator complex into a transcription factory which may be analogous to a liquid-liquid phase-separated condensate. The broad epigenetic domain has a dynamic chromatin structure which supports frequent transcription bursts. In this review, we present the current knowledge of broad epigenetic domains.

Mitogen-induced transcriptional programming in human fibroblasts.

Treatment of serum-starved quiescent human cells with fetal bovine serum (FBS), epidermal growth factor (EGF), or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) activates the RAS-MAPK pathway which initiates a transcriptional program which drives cells toward proliferation. Stimulation of the RAS-MAPK pathway activates mitogen- and stress-activated kinases (MSK) 1 and 2, which phosphorylate histone H3 at S10 (H3S10ph) or S28 (H3S28ph) (nucleosomal response) located at the regulatory regions of immediate-early genes, setting in motion a series of chromatin remodeling events that result in transcription initiation. To investigate immediate-early genes regulated by the MSK, we have completed transcriptome analyses (RNA sequencing) of human normal fibroblast cells (CCD-1070Sk) stimulated with EGF or TPA ± H89, a potent MSK/PKA inhibitor. The induction of many immediate-early genes was independent of MSK activity. However, the induction of immediate-early genes attenuated with H89 also had reduced induction with the PKA inhibitor, Rp-cAMPS. Several EGF-induced genes, coding for transcriptional repressors, were further upregulated with H89 but not with Rp-cAMPS, suggesting a role for MSK in modulating the induction level of these genes.

Transcriptionally Active Chromatin-Lessons Learned from the Chicken Erythrocyte Chromatin Fractionation.

The chicken erythrocyte model system has been valuable to the study of chromatin structure and function, specifically for genes involved in oxygen transport and the innate immune response. Several seminal features of transcriptionally active chromatin were discovered in this system. Davie and colleagues capitalized on the unique features of the chicken erythrocyte to separate and isolate transcriptionally active chromatin and silenced chromatin, using a powerful native fractionation procedure. Histone modifications, histone variants, atypical nucleosomes (U-shaped nucleosomes) and other chromatin structural features (open chromatin) were identified in these studies. More recently, the transcriptionally active chromosomal domains in the chicken erythrocyte genome were mapped by combining this chromatin fractionation method with next-generation DNA and RNA sequencing. The landscape of histone modifications relative to chromatin structural features in the chicken erythrocyte genome was reported in detail, including the first ever mapping of histone H4 asymmetrically dimethylated at Arg 3 (H4R3me2a) and histone H3 symmetrically dimethylated at Arg 2 (H3R2me2s), which are products of protein arginine methyltransferases (PRMTs) 1 and 5, respectively. PRMT1 is important in the establishment and maintenance of chicken erythrocyte transcriptionally active chromatin.

The treatment of SARS-CoV2 with antivirals and mitigation of the cytokine storm syndrome: the role of gene expression.

In the absence of a vaccine, the treatment of SARS-CoV2 has focused on eliminating the virus with antivirals or mitigating the cytokine storm syndrome (CSS) that leads to the most common cause of death: respiratory failure. Herein we discuss the mechanisms of antiviral treatments for SARS-CoV2 and treatment strategies for the CSS. Antivirals that have shown in vitro activity against SARS-CoV2, or the closely related SARS-CoV1 and MERS-CoV, are compared on the enzymatic level and by potency in cells. For treatment of the CSS, we discuss medications that reduce the effects or expression of cytokines involved in the CSS with an emphasis on those that reduce IL-6 because of its central role in the development of the CSS. We show that some of the medications covered influence the activity or expression of enzymes involved in epigenetic processes and specifically those that add or remove modifications to histones or DNA. Where available, the latest clinical data showing the efficacy of the medications is presented. With respect to their mechanisms, we explain why some medications are successful, why others have failed, and why some untested medications may yet prove useful.

The chicken model organism for epigenomic research.

The chicken model organism has advanced the areas of developmental biology, virology, immunology, oncology, epigenetic regulation of gene expression, conservation biology, and genomics of domestication. Further, the chicken model organism has aided in our understanding of human disease. Through the recent advances in high-throughput sequencing and bioinformatic tools, researchers have successfully identified sequences in the chicken genome that have human orthologs, improving mammalian genome annotation. In this review, we highlight the importance of chicken as an animal model in basic and pre-clinical research. We will present the importance of chicken in poultry epigenetics and in genomic studies that trace back to their ancestor, the last link between human and chicken in the tree of life. There are still many genes of unknown function in the chicken genome yet to be characterized. By taking advantage of recent sequencing technologies, it is possible to gain further insight into the chicken epigenome.

Genomic landscape of transcriptionally active histone arginine methylation marks, H3R2me2s and H4R3me2a, relative to nucleosome depleted regions.

Protein arginine methyltransferase 1 (PRMT1) and the product of this enzyme (histone H4 asymmetrically dimethylated at Arg 3; H4R3me2a) are important in the establishment and maintenance of chicken and murine erythrocyte transcriptionally active chromatin. Silencing the expression of PRMT1 results in loss of acetylated histones H3 and H4 and methylated H3K4 and prevents erythropoiesis. Here, we show that H4R3me2a and the PRMT5-catalyzed histone H3 symmetrically dimethylated at Arg 2 (H3R2me2s) locate largely to introns of expressed genes and intergenic regions, with both marks co-localizing in the chicken polychromatic erythrocyte genome. H4R3me2a and H3R2me2s were associated with histone marks of active promoters and enhancers, as well as with the body of genes that have an atypical chromatin structure, with nucleosome depleted regions. H4R3me2a co-localized with acetylated H3K27. Previous studies have shown that PRMT1 was bound to CBP/p300, suggesting a role of PRMT1-mediated H4R3me2a in CBP/p300 recruitment and H3K27 acetylation. Moreover, PRMT1 might be a key enzyme affected when S-adenosyl methionine levels are reduced in metabolic disorders.

Mitogen and stress- activated protein kinase regulated gene expression in cancer cells.

The mitogen- and stress-activated protein kinases activated by the extracellular-signal-regulated kinase 1/2 and/or stress-activated protein kinase 2/p38 mitogen-activated protein kinase pathways are recruited to the regulatory region of a subset of genes termed immediate-early genes, often leading to their induction. These genes, many of which code for transcription factors, have been directly linked to the phenotypic events in carcinogenesis. In this paper, we focus on the mitogen- and stress-activated protein kinases; their discovery, activation, H3 phosphorylation and recent discoveries in their roles in cancer.

Ubiquitin C-terminal hydrolase isozyme L1 is associated with shelterin complex at interstitial telomeric sites.

BACKGROUND: Ubiquitin C-terminal hydrolase isozyme L1 (UCHL1) is primarily expressed in neuronal cells and neuroendocrine cells and has been associated with various diseases, including many cancers. It is a multifunctional protein involved in deubiquitination, ubiquitination and ubiquitin homeostasis, but its specific roles are disputed and still generally undetermined. RESULTS: Herein, we demonstrate that UCHL1 is associated with genomic DNA in certain prostate cancer cell lines, including DU 145 cells derived from a brain metastatic site, and in HEK293T embryonic kidney cells with a neuronal lineage. Chromatin immunoprecipitation and sequencing revealed that UCHL1 localizes to TTAGGG repeats at telomeres and interstitial telomeric sequences, as do TRF1 and TRF2, components of the shelterin complex. A weak or transient interaction between UCHL1 and the shelterin complex was confirmed by immunoprecipitation and proximity ligation assays. UCHL1 and RAP1, also known as TERF2IP and a component of the shelterin complex, were bound to the nuclear scaffold. CONCLUSIONS: We demonstrated a novel feature of UCHL1 in binding telomeres and interstitial telomeric sites.

A 16 Yin Yang gene expression ratio signature for ER+/node- breast cancer.

Breast cancer is one of the leading causes of cancer death in women. It is a complex and heterogeneous disease with different clinical outcomes. Stratifying patients into subgroups with different outcomes could help guide clinical decision making. In this study, we used two opposing groups of genes, Yin and Yang, to develop a prognostic expression ratio signature. Using the METABRIC cohort we identified a16-gene signature capable of stratifying breast cancer patients into four risk levels with intention that low-risk patients would not undergo adjuvant systemic therapy, intermediate-low-risk patients will be treated with hormonal therapy only, and intermediate-high- and high-risk groups will be treated by chemotherapy in addition to the hormonal therapy. The 16-gene signature for four risk level stratifications of breast cancer patients has been validated using 14 independent datasets. Notably, the low-risk group (n = 51) of 205 estrogen receptor-positive and node negative (ER+/node-) patients from three different datasets who had not had any systemic adjuvant therapy had 100% 15-year disease-specific survival rate. The Concordance Index of YMR for ER+/node negative patients is close to the commercially available signatures. However, YMR showed more significance (HR = 3.7, p = 8.7e-12) in stratifying ER+/node- subgroup than OncotypeDx (HR = 2.7, p = 1.3e-7), MammaPrint (HR = 2.5, p = 5.8e-7), rorS (HR = 2.4, p = 1.4e-6), and NPI (HR = 2.6, p = 1.2e-6). YMR signature may be developed as a clinical tool to select a subgroup of low-risk ER+/node- patients who do not require any adjuvant hormonal therapy (AHT).

A 10-Gene Yin Yang Expression Ratio Signature for Stage IA and IB Non-Small Cell Lung Cancer.

INTRODUCTION: Lung cancer is the leading killer cancer worldwide. There is an urgent need for easy-to-use and robust clinical gene signatures for improved prognosis and treatment prediction. METHODS: We used a gene expression signature termed the Yin and Yang mean ratio (YMR), which is based on two groups of genes with opposing function, to determine lung cancer prognosis. The YMR signature represents the relative state of an individual tumor on a gene expression spectrum ranging from malignancy to the normal healthy lung. The genes in the YMR signature have therefore been determined independently of survival time, which is different from previous regression models. We then leveraged the cross-platform utility of the YMR signature to optimize the signature into a smaller set of genes that validated the robustness of the signature in many independent lung cancer expression data sets. RESULTS: Four Yin and six Yang genes were optimized using 741 NSCLC cases from diverse platforms, including microarray and RNA sequencing. The 10-gene signature demonstrated significant differences in survival in eight individual independent data sets and a larger combined 1346-patient data set. When multivariate analysis taking into account other common predictors of survival was used, the 5-year recurrence-free rate of YMR (p = 6.4 × 10-6, HR =1.71 [1.36-2.16]) was secondary only to stage. The YMR signature significantly separated high- and low-risk patients with stage IA or 1B adenocarcinoma and squamous cell carcinomas of all stages. The YMR signature can also predict the benefit of adjuvant chemotherapy in high-risk patients with stage I NSCLC. CONCLUSIONS: The YMR signature has great potential for guiding clinical management for NSCLC, particularly early-stage disease. The signature appears more reproducible than older signatures and functions using a variety of common gene expression platforms.

Dynamic Histone Acetylation of H3K4me3 Nucleosome Regulates MCL1 Pre-mRNA Splicing.

Pre-mRNA splicing is a cotranscriptional process affected by the chromatin architecture along the body of coding genes. Recruited to the pre-mRNA by splicing factors, histone deacetylases (HDACs) and K-acetyltransferases (KATs) catalyze dynamic histone acetylation along the gene. In colon carcinoma HCT 116 cells, HDAC inhibition specifically increased KAT2B occupancy as well as H3 and H4 acetylation of the H3K4 trimethylated (H3K4me3) nucleosome positioned over alternative exon 2 of the MCL1 gene, an event paralleled with the exclusion of exon 2. These results were reproduced in MDA-MB-231, but not in MCF7 breast adenocarcinoma cells. These later cells have much higher levels of demethylase KDM5B than either HCT 116 or MDA-MB-231 cells. We show that H3K4me3 steady-state levels and H3K4me3 occupancy at the end of exon 1 and over exon 2 of the MCL1 gene were lower in MCF7 than in MDA-MB-231 cells. Furthermore, in MCF7 cells, there was minimal effect of HDAC inhibition on H3/H4 acetylation and H3K4me3 levels along the MCL1 gene and no change in pre-mRNA splicing choice. These results show that, upon HDAC inhibition, the H3K4me3 mark plays a critical role in the exclusion of exon 2 from the MCL1 pre-mRNA. J. Cell. Physiol. 231: 2196-2204, 2016. © 2016 Wiley Periodicals, Inc.

High Mobility Group A2 protects cancer cells against telomere dysfunction.

The non-histone chromatin binding protein High Mobility Group AT-hook protein 2 (HMGA2) plays important roles in the repair and protection of genomic DNA in embryonic stem cells and cancer cells. Here we show that HMGA2 localizes to mammalian telomeres and enhances telomere stability in cancer cells. We present a novel interaction of HMGA2 with the key shelterin protein TRF2. We found that the linker (L1) region of HMGA2 contributes to this interaction but the ATI-L1-ATII molecular region of HMGA2 is required for strong interaction with TRF2. This interaction was independent of HMGA2 DNA-binding and did not require the TRF2 interacting partner RAP1 but involved the homodimerization and hinge regions of TRF2. HMGA2 retained TRF2 at telomeres and reduced telomere-dysfunction despite induced telomere stress. Silencing of HMGA2 resulted in (i) reduced binding of TRF2 to telomere DNA as observed by ChIP, (ii) increased telomere instability and (iii) the formation of telomere dysfunction-induced foci (TIF). This resulted in increased telomere aggregation, anaphase bridges and micronuclei. HMGA2 prevented ATM-dependent pTRF2T188 phosphorylation and attenuated signaling via the telomere specific ATM-CHK2-CDC25C DNA damage signaling axis. In summary, our data demonstrate a unique and novel role of HMGA2 in telomere protection and promoting telomere stability in cancer cells. This identifies HMGA2 as a new therapeutic target for the destabilization of telomeres in HMGA2+ cancer cells.

Connecting the dots: chromatin and alternative splicing in EMT.

Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.