Maintenance Of DNA Methylation Research Articles

Molecular pathogenesis of ICF syndrome

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is characterized by frequent appearance of multiradial chromosomes, which are distinctive chromosome fusions that occur at hypomethylated pericentromeric regions comprising repetitive sequences, in activated lymphocytes. The syndrome is caused by mutations in DNMT3B, ZBTB24, CDCA7, or HELLS. Dr. Motoko Unoki, Medical Institute of Bioregulation, Kyushu University, Japan, was involved in the team to identify the CDCA7 and HELLS genes as causative genes of ICF syndrome. Recent studies including Unoki's studies suggested that de novo DNA methylation is likely defective in patients with ICF syndrome harboring mutations in DNMT3B, whereas accumulating evidence suggests that replication-uncoupled maintenance DNA methylation of late-replicating regions is impaired in patients with ICF syndrome harboring mutations in ZBTB24, CDCA7, or HELLS. ZBTB24 is a transcriptional activator of CDCA7, and CDCA7 and HELLS compose a chromatin remodeling complex and are involved in the maintenance DNA methylation through an interaction with UHRF1 in a feed-forward manner. The latest study by Unoki possibly provided the missing link between DNA hypomethylation and the formation of the abnormal chromosomes; it could occur via aberrant transcription from the hypomethylated regions, followed by pathological R-loop formation. The homologous-recombination dominant condition caused by a defect in non-homologous end joining observed in several types of ICF syndrome could facilitate the formation of multiradial chromosomes.

High throughput screening identifies SOX2 as a super pioneer factor that inhibits DNA methylation maintenance at its binding sites

Binding of mammalian transcription factors (TFs) to regulatory regions is hindered by chromatin compaction and DNA methylation of their binding sites. Nevertheless, pioneer transcription factors (PFs), a distinct class of TFs, have the ability to access nucleosomal DNA, leading to nucleosome remodelling and enhanced chromatin accessibility. Whether PFs can bind to methylated sites and induce DNA demethylation is largely unknown. Using a highly parallelized approach to investigate PF ability to bind methylated DNA and induce DNA demethylation, we show that the interdependence between DNA methylation and TF binding is more complex than previously thought, even within a select group of TFs displaying pioneering activity; while some PFs do not affect the methylation status of their binding sites, we identified PFs that can protect DNA from methylation and others that can induce DNA demethylation at methylated binding sites. We call the latter super pioneer transcription factors (SPFs), as they are seemingly able to overcome several types of repressive epigenetic marks. Finally, while most SPFs induce TET-dependent active DNA demethylation, SOX2 binding leads to passive demethylation, an activity enhanced by the co-binding of OCT4. This finding suggests that SPFs could interfere with epigenetic memory during DNA replication.

Maintenance DNA methylation in pre-meiotic germ cells regulates meiotic prophase by facilitating homologous chromosome pairing.

Heterochromatin-related epigenetic mechanisms, such as DNA methylation, facilitate pairing of homologous chromosomes during the meiotic prophase of mammalian spermatogenesis. In pro-spermatogonia, de novo DNA methylation plays a key role in completing meiotic prophase and initiating meiotic division. However, the role of maintenance DNA methylation in the regulation of meiosis, especially in the adult, is not well understood. Here, we reveal that NP95 (also known as UHRF1) and DNMT1 - two essential proteins for maintenance DNA methylation - are co-expressed in spermatogonia and are necessary for meiosis in male germ cells. We find that Np95- or Dnmt1-deficient spermatocytes exhibit spermatogenic defects characterized by synaptic failure during meiotic prophase. In addition, assembly of pericentric heterochromatin clusters in early meiotic prophase, a phenomenon that is required for subsequent pairing of homologous chromosomes, is disrupted in both mutants. Based on these observations, we propose that DNA methylation, established in pre-meiotic spermatogonia, regulates synapsis of homologous chromosomes and, in turn, quality control of male germ cells. Maintenance DNA methylation, therefore, plays a role in ensuring faithful transmission of both genetic and epigenetic information to offspring.

Chromatin remodeling in replication-uncoupled maintenance DNA methylation and chromosome stability: Insights from ICF syndrome studies.

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is characterized by frequent appearance of multiradial chromosomes, which are distinctive chromosome fusions that occur at hypomethylated pericentromeric regions comprising repetitive sequences, in activated lymphocytes. The syndrome is caused by mutations in DNMT3B, ZBTB24, CDCA7, or HELLS. De novo DNA methylation is likely defective in patients with ICF syndrome harboring mutations in DNMT3B, whereas accumulating evidence suggests that replication‐uncoupled maintenance DNA methylation of late‐replicating regions is impaired in patients with ICF syndrome harboring mutations in ZBTB24, CDCA7, or HELLS. ZBTB24 is a transcriptional activator of CDCA7, and CDCA7 and HELLS compose a chromatin remodeling complex and are involved in the maintenance DNA methylation through an interaction with UHRF1 in a feed‐forward manner. Furthermore, our recent studies possibly provided the missing link between DNA hypomethylation and the formation of the abnormal chromosomes; it could occur via aberrant transcription from the hypomethylated regions, followed by pathological R‐loop formation. The homologous‐recombination dominant condition caused by a defect in nonhomologous end joining observed in several types of ICF syndrome could facilitate the formation of multiradial chromosomes. Here, the latest knowledge regarding maintenance DNA methylation and chromosome stability provided by those studies is reviewed.

Genome-wide identification and transcriptional characterization of DNA methyltransferases conferring temperature-sensitive male sterility in wheat

BackgroundDNA methyltransferase (DMT) genes contribute to plant stress responses and development by de novo establishment and subsequent maintenance of DNA methylation during replication. The photoperiod and/or temperature-sensitive genic male sterile (P/TGMS) lines play an important role in hybrid seed production of wheat. However, only a few studies have reported on the effect of DMT genes on temperature-sensitive male sterility of wheat. Although DMT genes have been investigated in some plant species, the identification and analysis of DMT genes in wheat (Triticum aestivum L.) based on genome-wide levels have not been reported.ResultsIn this study, a detailed overview of phylogeny of 52 wheat DMT (TaDMT) genes was presented. Homoeolog retention for TaDMT genes was significantly above the average retention rate for whole-wheat genes, indicating the functional importance of many DMT homoeologs. We found that the strikingly high number of TaDMT genes resulted mainly from the significant expansion of the TaDRM subfamily. Intriguingly, all 5 paralogs belonged to the wheat DRM subfamily, and we speculated that tandem duplications might play a crucial role in the TaDRM subfamily expansion. Through the transcriptional analysis of TaDMT genes in a TGMS line BS366 and its hybrids with the other six fertile lines under sterile and fertile conditions, we concluded that TaCMT-D2, TaMET1-B1, and TaDRM-U6 might be involved in male sterility in BS366. Furthermore, a correlation analysis showed that TaMET1-B1 might negatively regulate the expression of TaRAFTIN1A, an important gene for pollen development, so we speculated regarding an epigenetic regulatory mechanism underlying the male sterility of BS366 via the interaction between TaMET1-B1 and TaRAFTIN1A.ConclusionsOur findings presented a detailed phylogenic overview of the DMT genes and could provide novel insights into the effects of DMT genes on TGMS wheat.

Genome-wide impact of hydrogen peroxide on maintenance DNA methylation in replicating cells

BackgroundEnvironmental factors, such as oxidative stress, have the potential to modify the epigenetic landscape of cells. We have previously shown that DNA methyltransferase (DNMT) activity can be inhibited by sublethal doses of hydrogen peroxide (H2O2). However, site-specific changes in DNA methylation and the reversibility of any changes have not been explored. Using bead chip array technology, differential methylation was assessed in Jurkat T-lymphoma cells following exposure to H2O2.ResultsSublethal H2O2 exposure was associated with an initial genome-wide decrease in DNA methylation in replicating cells, which was largely corrected 72 h later. However, some alterations were conserved through subsequent cycles of cell division. Significant changes to the variability of DNA methylation were also observed both globally and at the site-specific level.ConclusionsThis research indicates that increased exposure to H2O2 can result in long-term alterations to DNA methylation patterns, providing a mechanism for environmental factors to have prolonged impact on gene expression.

PCNA-associated factor (KIAA0101/PCLAF) overexpression and gene copy number alterations in hepatocellular carcinoma tissues

BackgroundPCNA-associated factor, the protein encoded by the KIAA0101/PCLAF gene, is a cell-cycle regulated oncoprotein that regulates DNA synthesis, maintenance of DNA methylation, and DNA-damage bypass, through the interaction with the human sliding clamp PCNA. KIAA0101/PCLAF is overexpressed in various cancers, including hepatocellular carcinoma (HCC). However, it remains unknown whether KIAA0101/PCLAF overexpression is coupled to gene amplification in HCC.MethodsKIAA0101/PCLAF mRNA expression levels were assessed by quantitative real-time PCR (qRT-PCR) in 40 pairs of snap-frozen HCC and matched-non-cancerous tissues. KIAA0101/PCLAF gene copy numbers were evaluated by droplet digital PCR (ddPCR) in 36 pairs of the tissues, and protein expression was detected by immunohistochemistry (IHC) in 81 pairs of formalin-fixed paraffin-embedded (FFPE) tissues. The KIAA0101/PCLAF gene copy number alteration and RNA expression was compared by Spearman correlation. The relationships between KIAA0101 protein expression and other clinicopathological parameters, including Ki-67, p53, and HBsAg protein expression in HCC tissues, were evaluated using Chi-square test.ResultsOur results demonstrated that KIAA0101/PCLAF mRNA levels were significantly higher in HCC than in the matched-non-cancerous tissues (p < 0.0001). The high KIAA0101/PCLAF mRNA levels in HCC were associated with poor patient survival. The KIAA0101/PCLAF gene was not amplified in HCC, and KIAA0101/PCLAF gene copy numbers were not associated with KIAA0101/PCLAF transcript levels. KIAA0101 protein was overexpressed in the majority of HCC tissues (77.8%) but was not detectable in matched-non-cancerous tissues. Significant correlations between the expression of KIAA0101 protein in HCC tissues and p53 tumor suppressor protein (p = 0.002) and Ki-67 proliferation marker protein (p = 0.017) were found. However, KIAA0101 protein levels in HCC tissues were not correlated with patient age, tumor size, serum AFP level, or the HBsAg expression.ConclusionsKIAA0101/PCLAF mRNA and protein overexpression is frequently observed in HCC but without concurrent KIAA0101/PCLAF gene amplification. Significant correlations between the expression of KIAA0101 protein and p53 and Ki-67 proteins were observed in this study. Thus, detection of KIAA0101/PCLAF mRNA/protein might be used, along with the detection of p53 and Ki-67 proteins, as potential biomarkers to select candidate patients for further studies of novel HCC treatment related to these targets.

Humanization of a tandem repeat in IG-DMR causes stochastic restoration of paternal imprinting at mouse Dlk1-Dio3 domain.

The Dlk1-Dio3 imprinted domain, regulated by an intergenic differentially methylated region (IG-DMR), is important for mammalian embryonic development. Although previous studies have reported that DNA methylation of a tandem repeated array sequence in paternal IG-DMR (IG-DMR-Rep) plays an essential role in the maintenance of DNA methylation in mice, the function of a tandem repeated array sequence in human IG-DMR (hRep) is unknown. Here, we generated mice with a human tandem repeated sequence, which replaced the mouse IG-DMR-Rep. Mice that transmitted the humanized allele paternally exhibited variable methylation status at the IG-DMR and were stochastically rescued from the lethality of IG-DMR-Rep deficiency, suggesting that hRep plays a role in human IG-DMR for the regulation of imprinted expression. Moreover, chromatin immunoprecipitation analysis showed that TRIM28 was enriched in hypermethylated paternal hRep without ZFP57. Our results suggest that hRep contributes to the maintenance of human IG-DMR methylation imprints via the recruitment of TRIM28.

A novel class of selective non-nucleoside inhibitors of human DNA methyltransferase 3A

Screening of a small chemical library (Medicines for Malaria Venture Pathogen Box) identified two structurally related pyrazolone (inhibitor 1) and pyridazine (inhibitor 2) DNMT3A inhibitors with low micromolar inhibition constants. The uncompetitive and mixed type inhibition patterns with DNA and AdoMet suggest these molecules act through an allosteric mechanism, and thus are unlikely to bind to the enzyme’s active site. Unlike the clinically used mechanism based DNMT inhibitors such as decitabine or azacitidine that act via the enzyme active site, the inhibitors described here could lead to the development of more selective drugs. Both inhibitors show promising selectivity for DNMT3A in comparison to DNMT1 and bacterial DNA cytosine methyltransferases. With further study, this could form the basis of preferential targeting of de novo DNA methylation over maintenance DNA methylation.

Evidence for divergence of DNA methylation maintenance and a conserved inhibitory mechanism from DNA demethylation in chickens and mammals

BackgroundDNA methylation is a significant epigenetic modification that is evolutionarily conserved in various species and often serves as a repressive mark for transcription. DNA methylation levels and patterns are regulated by a balance of opposing enzyme functions, DNA methyltransferases, DNMT1/3A/3B and methylcytosine dioxygenases, TET1/2/3. In mice, the TET enzyme converts DNA cytosine methylation (5mC) to 5-hydroxymethylcytosine (5hmC) at the beginning of fertilisation and gastrulation and initiates a global loss of 5mC, while the 5mC level is increased on the onset of cell differentiation during early embryonic development.ObjectiveGlobal loss and gain of DNA methylation may be differently regulated in diverged species.MethodsChicken B-cell lymphoma DT40 cells were used as an avian model to compare differences in the overall regulation of DNA modification with mammals.ResultsWe found that DNA methylation is maintained at high levels in DT40 cells through compact chromatin formation, which inhibits TET-mediated demethylation. Human and mouse chromosomes introduced into DT40 cells by cell fusion lost the majority of 5mC, except for human subtelomeric repeats.ConclusionOur attempt to elucidate the differences in the epigenetic regulatory mechanisms between birds and mammals explored the evidence that they share a common chromatin-based regulation of TET–DNA access, while chicken DNMT1 is involved in different target sequence recognition systems, suggesting that factors inducing DNMT–DNA association have already diverged.

Distinguishing Active Versus Passive DNA Demethylation Using Illumina MethylationEPIC BeadChip Microarrays.

The 5-carbon positions on cytosine nucleotides preceding guanines in genomic DNA (CpG) are common targets for DNA methylation (5mC). DNA methylation removal can occur through both active and passive mechanisms. Ten-eleven translocation enzymes (TETs) oxidize 5mC in a stepwise manner to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5mC can also be removed passively through sequential cell divisions in the absence of DNA methylation maintenance. In this chapter, we describe approaches that couple TET-assisted bisulfite (TAB) and oxidative bisulfite (OxBS) conversion to the Illumina MethylationEPIC BeadChIP (EPIC array) and show how these technologies can be used to distinguish active versus passive DNA demethylation. We also describe integrative bioinformatics pipelines to facilitate this analysis.

Polycomb mutant partially suppresses DNA hypomethylation-associated phenotypes in Arabidopsis.

In plants and mammals, DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3), which is deposited by the polycomb repressive complex 2, are considered as two specialized systems for the epigenetic silencing of transposable element (TE) and genes, respectively. Nevertheless, many TE sequences acquire H3K27me3 when DNA methylation is lost. Here, we show in Arabidopsis thaliana that the gain of H3K27me3 observed at hundreds of TEs in the ddm1 mutant defective in the maintenance of DNA methylation, essentially depends on CURLY LEAF (CLF), one of two partially redundant H3K27 methyltransferases active in vegetative tissues. Surprisingly, the complete loss of H3K27me3 in ddm1 clf double mutant plants was not associated with further reactivation of TE expression nor with a burst of transposition. Instead, ddm1 clf plants exhibited less activated TEs, and a chromatin recompaction as well as hypermethylation of linker DNA compared with ddm1 Thus, a mutation in polycomb repressive complex 2 does not aggravate the molecular phenotypes linked to ddm1 but instead partially suppresses them, challenging our assumptions of the relationship between two conserved epigenetic silencing pathways.

In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.

Small DNA Methylation, Big Player in Plant Abiotic Stress Responses and Memory.

DNA methylation is a conserved epigenetic mark that plays important roles in maintaining genome stability and regulating gene expression. As sessile organisms, plants have evolved sophisticated regulatory systems to endure or respond to diverse adverse abiotic environmental challenges, i.e., abiotic stresses, such as extreme temperatures (cold and heat), drought and salinity. Plant stress responses are often accompanied by changes in chromatin modifications at diverse responsive loci, such as 5-methylcytosine (5mC) and N6-methyladenine (6mA) DNA methylation. Some abiotic stress responses are memorized for several hours or days through mitotic cell divisions and quickly reset to baseline levels after normal conditions are restored, which is referred to as somatic memory. In some cases, stress-induced chromatin marks are meiotically heritable and can impart the memory of stress exposure from parent plants to at least the next stress-free offspring generation through the mechanisms of transgenerational epigenetic inheritance, which may offer the descendants the potential to be adaptive for better fitness. In this review, we briefly summarize recent achievements regarding the establishment, maintenance and reset of DNA methylation, and highlight the diverse roles of DNA methylation in plant responses to abiotic stresses. Further, we discuss the potential role of DNA methylation in abiotic stress-induced somatic memory and transgenerational inheritance. Future research directions are proposed to develop stress-tolerant engineered crops to reduce the negative effects of abiotic stresses.

DNA maintenance methylation enzyme Dnmt1 in satellite cells is essential for muscle regeneration

Epigenetic transcriptional regulation is essential for the differentiation of various types of cells, including skeletal muscle cells. DNA methyltransferase 1 (Dnmt1) is responsible for maintenance of DNA methylation patterns via cell division. Here, we investigated the relationship between Dnmt1 and skeletal muscle regeneration. We found that Dnmt1 is upregulated in muscles during regeneration. To assess the role of Dnmt1 in satellite cells during regeneration, we performed conditional knockout (cKO) of Dnmt1 specifically in skeletal muscle satellite cells using Pax7CreERT2 mice and Dnmt1 flox mice. Muscle weight and the cross-sectional area after injury were significantly lower in Dnmt1 cKO mice than in control mice. RNA sequencing analysis revealed upregulation of genes involved in cell adhesion and apoptosis in satellite cells from cKO mice. Moreover, satellite cells cultured from cKO mice exhibited a reduced number of cells. These results suggest that Dnmt1 is an essential factor for muscle regeneration and is involved in positive regulation of satellite cell number.

Staying true to yourself: mechanisms of DNA methylation maintenance in mammals.

DNA methylation is essential to development and cellular physiology in mammals. Faulty DNA methylation is frequently observed in human diseases like cancer and neurological disorders. Molecularly, this epigenetic mark is linked to other chromatin modifications and it regulates key genomic processes, including transcription and splicing. Each round of DNA replication generates two hemi-methylated copies of the genome. These must be converted back to symmetrically methylated DNA before the next S-phase, or the mark will fade away; therefore the maintenance of DNA methylation is essential. Mechanistically, the maintenance of this epigenetic modification takes place during and after DNA replication, and occurs within the very dynamic context of chromatin re-assembly. Here, we review recent discoveries and unresolved questions regarding the mechanisms, dynamics and fidelity of DNA methylation maintenance in mammals. We also discuss how it could be regulated in normal development and misregulated in disease.

Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals

Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.

A role for LSH in facilitating DNA methylation by DNMT1 through enhancing UHRF1 chromatin association.

LSH, a SNF2 family DNA helicase, is a key regulator of DNA methylation in mammals. How LSH facilitates DNA methylation is not well defined. While previous studies with mouse embryonic stem cells (mESc) and fibroblasts (MEFs) derived from Lsh knockout mice have revealed a role of Lsh in de novo DNA methylation by Dnmt3a/3b, here we report that LSH contributes to DNA methylation in various cell lines primarily by promoting DNA methylation by DNMT1. We show that loss of LSH has a much bigger effect in DNA methylation than loss of DNMT3A and DNMT3B. Mechanistically, we demonstrate that LSH interacts with UHRF1 but not DNMT1 and facilitates UHRF1 chromatin association and UHRF1-catalyzed histone H3 ubiquitination in an ATPase activity-dependent manner, which in turn promotes DNMT1 recruitment to replication fork and DNA methylation. Notably, UHRF1 also enhances LSH association with the replication fork. Thus, our study identifies LSH as an essential factor for DNA methylation by DNMT1 and provides novel insight into how a feed-forward loop between LSH and UHRF1 facilitates DNMT1-mediated maintenance of DNA methylation in chromatin.

Maintenance DNA methylation is essential for regulatory T cell development and stability of suppressive function.

Tregs require Foxp3 expression and induction of a specific DNA hypomethylation signature during development, after which Tregs persist as a self-renewing population that regulates immune system activation. Whether maintenance DNA methylation is required for Treg lineage development and stability and how methylation patterns are maintained during lineage self-renewal remain unclear. Here, we demonstrate that the epigenetic regulator ubiquitin-like with plant homeodomain and RING finger domains 1 (Uhrf1) is essential for maintenance of methyl-DNA marks that stabilize Treg cellular identity by repressing effector T cell transcriptional programs. Constitutive and induced deficiency of Uhrf1 within Foxp3+ cells resulted in global yet nonuniform loss of DNA methylation, derepression of inflammatory transcriptional programs, destabilization of the Treg lineage, and spontaneous inflammation. These findings support a paradigm in which maintenance DNA methylation is required in distinct regions of the Treg genome for both lineage establishment and stability of identity and suppressive function.

The histone and non-histone methyllysine reader activities of the UHRF1 tandem Tudor domain are dispensable for the propagation of aberrant DNA methylation patterning in cancer cells

The chromatin-binding E3 ubiquitin ligase ubiquitin-like with PHD and RING finger domains 1 (UHRF1) contributes to the maintenance of aberrant DNA methylation patterning in cancer cells through multivalent histone and DNA recognition. The tandem Tudor domain (TTD) of UHRF1 is well-characterized as a reader of lysine 9 di- and tri-methylation on histone H3 (H3K9me2/me3) and, more recently, lysine 126 di- and tri-methylation on DNA ligase 1 (LIG1K126me2/me3). However, the functional significance and selectivity of these interactions remain unclear. In this study, we used protein domain microarrays to search for additional readers of LIG1K126me2, the preferred methyl state bound by the UHRF1 TTD. We show that the UHRF1 TTD binds LIG1K126me2 with high affinity and selectivity compared to other known methyllysine readers. Notably, and unlike H3K9me2/me3, the UHRF1 plant homeodomain (PHD) and its N-terminal linker (L2) do not contribute to multivalent LIG1K126me2 recognition along with the TTD. To test the functional significance of this interaction, we designed a LIG1K126me2 cell-penetrating peptide (CPP). Consistent with LIG1 knockdown, uptake of the CPP had no significant effect on the propagation of DNA methylation patterning across the genomes of bulk populations from high-resolution analysis of several cancer cell lines. Further, we did not detect significant changes in DNA methylation patterning from bulk cell populations after chemical or genetic disruption of lysine methyltransferase activity associated with LIG1K126me2 and H3K9me2. Collectively, these studies identify UHRF1 as a selective reader of LIG1K126me2 in vitro and further implicate the histone and non-histone methyllysine reader activity of the UHRF1 TTD as a dispensable domain function for cancer cell DNA methylation maintenance.