RT-DOb, a switch gene for the gene pair {Csf1r, Milr1}, can influence the onset of Alzheimer's disease by regulating communication between mast cell and microglia.

In biology, homeostasis is a central cellular phenomenon that plays a crucial role in survival. The central nervous system (CNS) is controlled by exquisitely sensitive homeostatic mechanisms when facing inflammatory or pathological insults. Mast cells and microglia play a crucial role in CNS homeostasis by eliminating damaged or unnecessary neurons and synapses. Therefore, decoding molecular circuits that regulate CNS homeostasis may lead to more effective therapeutic strategies that specifically target particular subsets for better therapy of Alzheimer's disease (AD). Based on a computational analysis of a microarray dataset related to AD, the H2-Ob gene was previously identified as a potential modulator of the homeostatic balance between mast cells and microglia. Specifically, it plays such a role in the presence of a three-way gene interaction in which the H2-Ob gene acts as a switch in the co-expression relationship of two genes, Csf1r and Milr1. Therefore, the importance of the H2-Ob gene as a potential therapeutic target for AD has led us to experimentally validate this relationship using the quantitative real-time PCR technique. In the experimental investigation, we confirmed that a change in the expression levels of the RT1-DOb gene (the rat ortholog of murine H2-Ob) can switch the co-expression relationship between Csf1r and Milr1. Furthermore, since the RT1-DOb gene is up-regulated in AD, the mentioned triplets might be related to triggering AD.

Loss of G0/G1 switch gene 2 (G0S2) promotes disease progression and drug resistance in chronic myeloid leukaemia (CML) by disrupting glycerophospholipid metabolism.

Tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1 have turned chronic myeloid leukaemia (CML) from a fatal disease into a manageable condition for most patients. Despite improved survival, targeting drug-resistant leukaemia stem cells (LSCs) remains a challenge for curative CML therapy. Aberrant lipid metabolism can have a large impact on membrane dynamics, cell survival and therapeutic responses in cancer. While ceramide and sphingolipid levels were previously correlated with TKI response in CML, the role of lipid metabolism in TKI resistance is not well understood. We have identified downregulation of a critical regulator of lipid metabolism, G0/G1 switch gene 2 (G0S2), in multiple scenarios of TKI resistance, including (1) BCR::ABL1 kinase-independent TKI resistance, (2) progression of CML from the chronic to the blast phase of the disease, and (3) in CML versus normal myeloid progenitors. Accordingly, CML patients with low G0S2 expression levels had a worse overall survival. G0S2 downregulation in CML was not a result of promoter hypermethylation or BCR::ABL1 kinase activity, but was rather due to transcriptional repression by MYC. Using CML cell lines, patient samples and G0s2 knockout (G0s2-/- ) mice, we demonstrate a tumour suppressor role for G0S2 in CML and TKI resistance. Our data suggest that reduced G0S2 protein expression in CML disrupts glycerophospholipid metabolism, correlating with a block of differentiation that renders CML cells resistant to therapy. Altogether, our data unravel a new role for G0S2 in regulating myeloid differentiation and TKI response in CML, and suggest that restoring G0S2 may have clinical utility.

Nkx3-1 and Fech genes might be switch genes involved in pituitary non-functioning adenoma invasiveness.

Non-functioning pituitary adenomas (NFPAs) are typical pituitary macroadenomas in adults associated with increased mortality and morbidity. Although pituitary adenomas are commonly considered slow-growing benign brain tumors, numerous of them possess an invasive nature. Such tumors destroy sella turcica and invade the adjacent tissues such as the cavernous sinus and sphenoid sinus. In these cases, the most critical obstacle for complete surgical removal is the high risk of damaging adjacent vital structures. Therefore, the development of novel therapeutic strategies for either early diagnosis through biomarkers or medical therapies to reduce the recurrence rate of NFPAs is imperative. Identification of gene interactions has paved the way for decoding complex molecular mechanisms, including disease-related pathways, and identifying the most momentous genes involved in a specific disease. Currently, our knowledge of the invasion of the pituitary adenoma at the molecular level is not sufficient. The current study aimed to identify critical biomarkers and biological pathways associated with invasiveness in the NFPAs using a three-way interaction model for the first time. In the current study, the Liquid association method was applied to capture the statistically significant triplets involved in NFPAs invasiveness. Subsequently, Random Forest analysis was applied to select the most important switch genes. Finally, gene set enrichment (GSE) and gene regulatory network (GRN) analyses were applied to trace the biological relevance of the statistically significant triplets. The results of this study suggest that "mRNA processing" and "spindle organization" biological processes are important in NFAPs invasiveness. Specifically, our results suggest Nkx3-1 and Fech as two switch genes in NFAPs invasiveness that may be potential biomarkers or target genes in this pathology.

Emerging mammalian gene switches for controlling implantable cell therapies.

Engineered cell-based therapies have emerged as a new paradigm in modern medicine, with several engineered T cell therapies currently approved to treat blood cancers and many more in clinical development. Tremendous progress in synthetic biology over the past two decades has allowed us to program cells with sophisticated sense-and-response modules that can effectively control therapeutic functions. In this review, we highlight recent advances in mammalian synthetic gene switches, focusing on devices designed for therapeutic applications. Although many gene switches responding to endogenous or exogenous molecular signals have been developed, the focus is shifting towards achieving remote-controlled production of therapeutic effectors by stimulating implanted engineered cells with traceless physical signals, such as light, electrical signals, magnetic fields, heat or ultrasound.

Exploring polyps to colon carcinoma voyage: can blocking the crossroad halt the sequence?

Colorectal cancer is an important public health concern leading to significant cancer associate mortality. A vast majority of colon cancer arises from polyp which later follows adenoma, adenocarcinoma, and carcinoma sequence. This whole process takes several years to complete and recent genomic and proteomic technologies are identifying several targets involved in each step of polyp to carcinoma transformation in a large number of studies. Current text presents interaction network of targets involved in polyp to carcinoma transformation. In addition, important targets involved in each step according to network biological parameters are also presented. The functional overrepresentation analysis of each step targets and common top biological processes and pathways involved in carcinoma indicate several insights about this whole mechanism. Interaction networks indicate TP53, AKT1, GAPDH, INS, EGFR, and ALB as the most important targets commonly involved in polyp to carcinoma sequence. Though several important pathways are known to be involved in CRC, the central common involvement of PI3K-AKT indicates its potential for devising CRC management strategies. The common and central targets and pathways involved in polyp to carcinoma progression can shed light on its mechanism and potential management strategies. The data-driven approach aims to add valuable inputs to the mechanism of the years-long polyp-carcinoma sequence.

Key Disease Mechanisms Linked to Alzheimer's Disease in the Entorhinal Cortex.

Alzheimer's disease (AD) is a chronic, neurodegenerative brain disorder affecting millions of Americans that is expected to increase in incidence with the expanding aging population. Symptomatic AD patients show cognitive decline and often develop neuropsychiatric symptoms due to the accumulation of insoluble proteins that produce plaques and tangles seen in the brain at autopsy. Unexpectedly, some clinically normal individuals also show AD pathology in the brain at autopsy (asymptomatic AD, AsymAD). In this study, SWItchMiner software was used to identify key switch genes in the brain's entorhinal cortex that lead to the development of AD or disease resilience. Seventy-two switch genes were identified that are differentially expressed in AD patients compared to healthy controls. These genes are involved in inflammation, platelet activation, and phospholipase D and estrogen signaling. Peroxisome proliferator-activated receptor γ (PPARG), zinc-finger transcription factor (YY1), sterol regulatory element-binding transcription factor 2 (SREBF2), and early growth response 1 (EGR1) were identified as transcription factors that potentially regulate switch genes in AD. Comparing AD patients to AsymAD individuals revealed 51 switch genes; PPARG as a potential regulator of these genes, and platelet activation and phospholipase D as critical signaling pathways. Chemical-protein interaction analysis revealed that valproic acid is a therapeutic agent that could prevent AD from progressing.

A Wnt-mediated phenotype switch along the epithelial-mesenchymal axis defines resistance and invasion downstream of ionising radiation in oral squamous cell carcinoma.

BACKGROUND: Dynamic transitions of tumour cells along the epithelial-mesenchymal axis are important in tumorigenesis, metastasis and therapy resistance. METHODS: In this study, we have used cell lines, 3D spheroids and tumour samples in a variety of cell biological and transcriptome analyses to highlight the cellular and molecular dynamics of OSCC response to ionising radiation. RESULTS: Our study demonstrates a prominent hybrid epithelial-mesenchymal state in oral squamous cell carcinoma cells and tumour samples. We have further identified a key role for levels of E-cadherin in stratifying the hybrid cells to compartments with varying levels of radiation response and radiation-induced epithelial-mesenchymal transition. The response to radiation further entailed the generation of a new cell population with low expression levels of E-cadherin, and positive for Vimentin (ECADLow/Neg-VIMPos), a phenotypic signature that showed an enhanced capacity for radiation resistance and invasion. At the molecular level, transcriptome analysis of spheroids in response to radiation showed an initial burst of misregulation within the first 30 min that further declined, although still highlighting key alterations in gene signatures. Among others, pathway analysis showed an over-representation for the Wnt signalling pathway that was further confirmed to be functionally involved in the generation of ECADLow/Neg-VIMPos population, acting upstream of radiation resistance and tumour cell invasion. CONCLUSION: This study highlights the functional significance and complexity of tumour cell remodelling in response to ionising radiation with links to resistance and invasive capacity. An area of less focus in conventional radiotherapy, with the potential to improve treatment outcomes and relapse-free survival.

Molecular switch from MYC to MYCN expression in MYC protein negative Burkitt lymphoma cases.

MYC is the most altered oncogene in human cancer, and belongs to a large family of genes, including MYCN and MYCL. Recently, while assessing the degree of correlation between MYC gene rearrangement and MYC protein expression in aggressive B-cell lymphomas, we observed few Burkitt lymphoma (BL) cases lacking MYC protein expression despite the translocation involving the MYC gene. Therefore, in the present study we aimed to better characterize such cases. Our results identified two sub-groups of MYC protein negative BL: one lacking detectable MYC protein expression but presenting MYCN mRNA and protein expression; the second characterized by the lack of both MYC and MYCN proteins but showing MYC mRNA. Interestingly, the two sub-groups presented a different pattern of SNVs affecting MYC gene family members that may induce the switch from MYC to MYCN. Particulary, MYCN-expressing cases show MYCN SNVs at interaction interface that stabilize the protein associated with loss-of-function of MYC. This finding highlights MYCN as a reliable diagnostic marker in such cases. Nevertheless, due to the overlapping clinic, morphology and immunohistochemistry (apart for MYC versus MYCN protein expression) of both sub-groups, the described cases represent bona fide BL according to the current criteria of the World Health Organization.

The E-Cadherin and N-Cadherin Switch in Epithelial-to-Mesenchymal Transition: Signaling, Therapeutic Implications, and Challenges.

Epithelial-to-Mesenchymal Transition (EMT) has been shown to be crucial in tumorigenesis where the EMT program enhances metastasis, chemoresistance and tumor stemness. Due to its emerging role as a pivotal driver of tumorigenesis, targeting EMT is of great therapeutic interest in counteracting metastasis and chemoresistance in cancer patients. The hallmark of EMT is the upregulation of N-cadherin followed by the downregulation of E-cadherin, and this process is regulated by a complex network of signaling pathways and transcription factors. In this review, we summarized the recent understanding of the roles of E- and N-cadherins in cancer invasion and metastasis as well as the crosstalk with other signaling pathways involved in EMT. We also highlighted a few natural compounds with potential anti-EMT property and outlined the future directions in the development of novel intervention in human cancer treatments. We have reviewed 287 published papers related to this topic and identified some of the challenges faced in translating the discovery work from bench to bedside.

The Roles of Hippo Signaling Transducers Yap and Taz in Chromatin Remodeling.

Hippo signaling controls cellular processes that ultimately impact organogenesis and homeostasis. Consequently, disease states including cancer can emerge when signaling is deregulated. The major pathway transducers Yap and Taz require cofactors to impart transcriptional control over target genes. Research into Yap/Taz-mediated epigenetic modifications has revealed their association with chromatin-remodeling complex proteins as a means of altering chromatin structure, therefore affecting accessibility and activity of target genes. Specifically, Yap/Taz have been found to associate with factors of the GAGA, Ncoa6, Mediator, Switch/sucrose nonfermentable (SWI/SNF), and Nucleosome Remodeling and Deacetylase (NuRD) chromatin-remodeling complexes to alter the accessibility of target genes. This review highlights the different mechanisms by which Yap/Taz collaborate with other factors to modify DNA packing at specific loci to either activate or repress target gene transcription.

HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma.

The mechanisms by which regulatory T cells (Tregs) migrate to and function within the hypoxic tumor microenvironment are unclear. Our studies indicate that specific ablation of hypoxia-inducible factor 1α (HIF-1α) in Tregs results in enhanced CD8+ T cell suppression versus wild-type Tregs under hypoxia, due to increased pyruvate import into the mitochondria. Importantly, HIF-1α-deficient Tregs are minimally affected by the inhibition of lipid oxidation, a fuel that is critical for Treg metabolism in tumors. Under hypoxia, HIF-1α directs glucose away from mitochondria, leaving Tregs dependent on fatty acids for mitochondrial metabolism within the hypoxic tumor. Indeed, inhibition of lipid oxidation enhances the survival of mice with glioma. Interestingly, HIF-1α-deficient-Treg mice exhibit significantly enhanced animal survival in a murine model of glioma, due to their stymied migratory capacity, explaining their reduced abundance in tumor-bearing mice. Thus HIF-1α acts as a metabolic switch for Tregs between glycolytic-driven migration and oxidative phosphorylation-driven immunosuppression.

SWIM tool application to expression data of glioblastoma stem-like cell lines, corresponding primary tumors and conventional glioma cell lines.

BACKGROUND: It is well-known that glioblastoma contains self-renewing, stem-like subpopulation with the ability to sustain tumor growth. These cells - called cancer stem-like cells - share certain phenotypic characteristics with untransformed stem cells and are resistant to many conventional cancer therapies, which might explain the limitations in curing human malignancies. Thus, the identification of genes controlling the differentiation of these stem-like cells is becoming a successful therapeutic strategy, owing to the promise of novel targets for treating malignancies. METHODS: Recently, we developed SWIM, a software able to unveil a small pool of genes - called switch genes - critically associated with drastic changes in cell phenotype. Here, we applied SWIM to the expression profiling of glioblastoma stem-like cells and conventional glioma cell lines, in order to identify switch genes related to stem-like phenotype. RESULTS: SWIM identifies 171 switch genes that are all down-regulated in glioblastoma stem-like cells. This list encompasses genes like CAV1, COL5A1, COL6A3, FLNB, HMMR, ITGA3, ITGA5, MET, SDC1, THBS1, and VEGFC, involved in "ECM-receptor interaction" and "focal adhesion" pathways. The inhibition of switch genes highly correlates with the activation of genes related to neural development and differentiation, such as the 4-core OLIG2, POU3F2, SALL2, SOX2, whose induction has been shown to be sufficient to reprogram differentiated glioblastoma into stem-like cells. Among switch genes, the transcription factor FOSL1 appears as the brightest star since: it is down-regulated in stem-like cells; it highly negatively correlates with the 4-core genes that are all up-regulated in stem-like cells; the promoter regions of the 4-core genes harbor a consensus binding motif for FOSL1. CONCLUSIONS: We suggest that the inhibition of switch genes in stem-like cells could induce the deregulation of cell communication pathways, contributing to neoplastic progression and tumor invasiveness. Conversely, their activation could restore the physiological equilibrium between cell adhesion and migration, hampering the progression of cancer. Moreover, we posit FOSL1 as promising candidate to orchestrate the differentiation of cancer stem-like cells by repressing the 4-core genes' expression, which severely halts cancer growth and might affect the therapeutic outcome. We suggest FOSL1 as novel putative therapeutic and prognostic biomarker, worthy of further investigation.

Transcriptome Analyses of Female Somatotropes and Lactotropes Reveal Novel Regulators of Cell Identity in the Pituitary.

The differentiation of the hormone-producing cell lineages of the anterior pituitary represents an informative model of mammalian cell fate determination. The generation and maintenance of two of these lineages, the GH-producing somatotropes and prolactin (PRL)-producing lactotropes, are dependent on the pituitary-specific transcription factor POU1F1. Whereas POU1F1 is expressed in both cell types, and plays a direct role in the activation of both the Gh and Prl genes, GH expression is restricted to somatotropes and PRL expression is restricted to lactotropes. These observations imply the existence of additional, cell type-enriched factors that contribute to the somatotrope and lactotrope cell identities. In this study, we use transgenic mouse models to facilitate sorting of somatotrope and lactotrope populations based on the expression of fluorescent markers expressed under Gh and Prl gene transcriptional controls. The transcriptomic analyses reveal a concordance of gene expression profiles in the two populations. The limited number of divergent mRNAs between the two populations includes a set of transcription factors that may have roles in pituitary lineage divergence and/or in regulating expression of cell type-specific genes after differentiation. Four of these factors were validated for lineage enrichment at the level of protein expression, two somatotrope enriched and two lactotrope enriched. Three of these four factors were shown to have corresponding activities in appropriate enhancement or repression of landmark genes in a cell culture model system. These studies identify novel regulators of the somatotropes and lactotropes, and they establish a useful database for further study of these lineages in the anterior pituitary.

Identification of a Novel Enhancer/Chromatin Opening Element Associated with High-Level γ-Globin Gene Expression.

The organization of the five ß-type globin genes on chromosome 11 reflects the timing of expression during erythroid cell development, with the embryonic ε-globin gene being located at the 5' end, followed by the two fetal γ-globin genes, and with the adult ß- and δ-globin genes being located at the 3' end. Here, we functionally characterized a DNase I-hypersensitive site (HS) located 4 kb upstream of the Gγ-globin gene (HBG-4kb HS). This site is occupied by transcription factors USF1, USF2, EGR1, MafK, and NF-E2 in the human erythroleukemia cell line K562 and exhibits histone modifications typical for enhancers. We generated a synthetic zinc finger (ZF) DNA-binding domain targeting the HBG-4kb HS (HBG-4kb ZF). The HBG-4kb ZF interacted with the target site in vitro and in the context of cells with a high affinity and specificity. Direct delivery of the HBG-4kb ZF to K562 and primary human erythroid cells caused a reduction in γ-globin gene expression which was associated with decreased binding of transcription factors and active histone marks at and downstream of the HS. The data demonstrate that the HBG-4kb HS is important for fetal globin production and suggest that it may act by opening chromatin in a directional manner.

DNA Binding Induces a Nanomechanical Switch in the RRM1 Domain of TDP-43.

Understanding the molecular mechanisms governing protein-nucleic acid interactions is fundamental to many nuclear processes. However, how nucleic acid binding affects the conformation and dynamics of the substrate protein remains poorly understood. Here we use a combination of single molecule force spectroscopy AFM and biochemical assays to show that the binding of TG-rich ssDNA triggers a mechanical switch in the RRM1 domain of TDP-43, toggling between an entropic spring devoid of mechanical stability and a shock absorber bound-form that resists unfolding forces of ∼40 pN. The fraction of mechanically resistant proteins correlates with an increasing length of the TG n oligonucleotide, demonstrating that protein mechanical stability is a direct reporter of nucleic acid binding. Steered molecular dynamics simulations on related RNA oligonucleotides reveal that the increased mechanical stability fingerprinting the holo-form is likely to stem from a unique scenario whereby the nucleic acid acts as a "mechanical staple" that protects RRM1 from mechanical unfolding. Our approach highlights nucleic acid binding as an effective strategy to control protein nanomechanics.

Genetic and Epigenetic Mechanisms of ß-Globin Gene Switching.

Vertebrates have multiple forms of hemoglobin that differ in the composition of their polypeptide chains. During ontogenesis, the composition of these subunits changes. Genes encoding different α- and ß-polypeptide chains are located in two multigene clusters on different chromosomes. Each cluster contains several genes that are expressed at different stages of ontogenesis. The phenomenon of stage-specific transcription of globin genes is referred to as globin gene switching. Mechanisms of expression switching, stage-specific activation, and repression of transcription of α- and ß-globin genes are of interest from both theoretical and practical points of view. Alteration of balanced expression of globin genes, which usually occurs due to damage to adult ß-globin genes, leads to development of severe diseases - hemoglobinopathies. In most cases, reactivation of the fetal hemoglobin gene in patients with ß-thalassemia and sickle cell disease can reduce negative consequences of irreversible alterations of expression of the ß-globin genes. This review focuses on the current state of research on genetic and epigenetic mechanisms underlying stage-specific switching of ß-globin genes.

A Switchable Site-Specific Antibody Conjugate.

Genetic incorporation of unnatural amino acids (UAAs) provides a unique approach to the synthesis of site-specific antibody conjugates that are homogeneous and better defined constructs than random conjugates. Yet, the yield varies for every antibody, and the process is costly and time-consuming. We have developed a switchable αGCN4-Fab conjugate that incorporates UAA p-acetylphenylalanine. The GCN4 peptide is used as a switch, and antibodies fused by GCN4 can direct the αGCN4-Fab conjugate to target different cancer cells for diagnosis, imaging, or therapeutic treatment. More importantly, this switchable conjugate demonstrated an impressive potential for pretargeted imaging in vivo. This approach illustrates the utility of an orthogonal switch as a general strategy to endow versatility to a single antibody conjugate, which should facilitate the application of UAA-based site-specific conjugates for a host of biomedical uses in the future.

Role of Complex Epigenetic Switching in Tumor Necrosis Factor-α Upregulation in the Prefrontal Cortex of Suicide Subjects.

OBJECTIVE: Proinflammatory cytokines have recently received considerable attention for their role in suicidal behavior; however, how the expression of cytokine genes is regulated is not clearly known. The authors examined underlying mechanisms of critical cytokine gene tumor necrosis factor-alpha (TNF-α) dysregulation in the brains of individuals who died by suicide. METHOD: TNF-α expression was examined in the dorsolateral prefrontal cortex of the postmortem brains of persons with and without major depressive disorder who died by suicide and of persons with major depressive disorder who died of causes other than suicide. The role of putative microRNAs targeting TNF-α and RNA-binding protein Hu antigen R (HuR) was tested with in vitro and in vivo approaches and by examining expression of transactivation response RNA binding protein (TRBP). Genetic influence on TNF-α expression was determined by expression quantitative trait loci analysis and by genotyping three single-nucleotide polymorphisms in the promoter region of the TNF-α gene. Promoter methylation of TNF-α was determined by using methylated DNA immunoprecipitation assay. Expression of miR-19a-3p and TNF-α was also determined in the peripheral blood mononuclear cells of 12 healthy control subjects and 12 currently depressed patients with severe suicidal ideation. RESULTS: TNF-α expression was significantly higher in the dorsolateral prefrontal cortex of individuals who died by suicide, regardless of psychiatric diagnosis. Its expression level was also increased in individuals with major depressive disorder who died by causes other than suicide. On the other hand, expression of miR-19a-3p was upregulated specifically in individuals who died by suicide. In a preliminary observation, similar upregulation of TNF-α and miR-19a-3p was observed in the peripheral blood mononuclear cells of depressed patients with suicidal ideation. Despite its ability to directly target TNF-α in vitro, miR-19a-3p showed no interaction with TNF-α in the dorsolateral prefrontal cortex. HuR potentially stabilized TNF-α transcript, presumably by sequestering its 3' untranslated region from miR-19a-3p-mediated inhibition. Furthermore, decreased TRBP expression supported abnormality in the interaction between miR-19a-3p and TNF-α. Additionally, TNF-α transcriptional upregulation was associated with promoter hypomethylation, whereas no genetic influence on altered TNF-α or miR-19a-3p expression was observed in individuals who died by suicide. CONCLUSIONS: The data in this study provide mechanistic insights into the dysregulation of the TNF-α gene in the brains of individuals who died by suicide, which could potentially be involved in suicidal behavior.

Genetic determinants and epigenetic effects of pioneer-factor occupancy.

Transcription factors (TFs) direct developmental transitions by binding to target DNA sequences, influencing gene expression and establishing complex gene-regultory networks. To systematically determine the molecular components that enable or constrain TF activity, we investigated the genomic occupancy of FOXA2, GATA4 and OCT4 in several cell types. Despite their classification as pioneer factors, all three TFs exhibit cell-type-specific binding, even when supraphysiologically and ectopically expressed. However, FOXA2 and GATA4 can be distinguished by low enrichment at loci that are highly occupied by these factors in alternative cell types. We find that expression of additional cofactors increases enrichment at a subset of these sites. Finally, FOXA2 occupancy and changes to DNA accessibility can occur in G1-arrested cells, but subsequent loss of DNA methylation requires DNA replication.