Maintenance DNA Methyltransferase DNMT1 Research Articles

Coevolution of the CDCA7-HELLS ICF-related nucleosome remodeling complex and DNA methyltransferases

5-Methylcytosine (5mC) and DNA methyltransferases (DNMTs) are broadly conserved in eukaryotes but are also frequently lost during evolution. The mammalian SNF2 family ATPase HELLS and its plant ortholog DDM1 are critical for maintaining 5mC. Mutations in HELLS, its activator CDCA7, and the de novo DNA methyltransferase DNMT3B, cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, a genetic disorder associated with the loss of DNA methylation. We here examine the coevolution of CDCA7, HELLS and DNMTs. While DNMT3, the maintenance DNA methyltransferase DNMT1, HELLS, and CDCA7 are all highly conserved in vertebrates and green plants, they are frequently co-lost in other evolutionary clades. The presence-absence patterns of these genes are not random; almost all CDCA7 harboring eukaryote species also have HELLS and DNMT1 (or another maintenance methyltransferase, DNMT5). Coevolution of presence-absence patterns (CoPAP) analysis in Ecdysozoa further indicates coevolutionary linkages among CDCA7, HELLS, DNMT1 and its activator UHRF1. We hypothesize that CDCA7 becomes dispensable in species that lost HELLS or DNA methylation, and/or the loss of CDCA7 triggers the replacement of DNA methylation by other chromatin regulation mechanisms. Our study suggests that a unique specialized role of CDCA7 in HELLS-dependent DNA methylation maintenance is broadly inherited from the last eukaryotic common ancestor.

Coevolution of the CDCA7-HELLS ICF-related nucleosome remodeling complex and DNA methyltransferases.

5-Methylcytosine (5mC) and DNA methyltransferases (DNMTs) are broadly conserved in eukaryotes but are also frequently lost during evolution. The mammalian SNF2 family ATPase HELLS and its plant ortholog DDM1 are critical for maintaining 5mC. Mutations in HELLS, its activator CDCA7, and the de novo DNA methyltransferase DNMT3B, cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, a genetic disorder associated with the loss of DNA methylation. We here examine the coevolution of CDCA7, HELLS and DNMTs. While DNMT3, the maintenance DNA methyltransferase DNMT1, HELLS, and CDCA7 are all highly conserved in vertebrates and green plants, they are frequently co-lost in other evolutionary clades. The presence-absence patterns of these genes are not random; almost all CDCA7 harboring eukaryote species also have HELLS and DNMT1 (or another maintenance methyltransferase, DNMT5). Coevolution of presence-absence patterns (CoPAP) analysis in Ecdysozoa further indicates coevolutionary linkages among CDCA7, HELLS, DNMT1 and its activator UHRF1. We hypothesize that CDCA7 becomes dispensable in species that lost HELLS or DNA methylation, and/or the loss of CDCA7 triggers the replacement of DNA methylation by other chromatin regulation mechanisms. Our study suggests that a unique specialized role of CDCA7 in HELLS-dependent DNA methylation maintenance is broadly inherited from the last eukaryotic common ancestor.

DNMT1 mutant ants develop normally but have disrupted oogenesis

Although DNA methylation is an important gene regulatory mechanism in mammals, its function in arthropods remains poorly understood. Studies in eusocial insects have argued for its role in caste development by regulating gene expression and splicing. However, such findings are not always consistent across studies, and have therefore remained controversial. Here we use CRISPR/Cas9 to mutate the maintenance DNA methyltransferase DNMT1 in the clonal raider ant, Ooceraea biroi. Mutants have greatly reduced DNA methylation, but no obvious developmental phenotypes, demonstrating that, unlike mammals, ants can undergo normal development without DNMT1 or DNA methylation. Additionally, we find no evidence of DNA methylation regulating caste development. However, mutants are sterile, whereas in wild-type ants, DNMT1 is localized to the ovaries and maternally provisioned into nascent oocytes. This supports the idea that DNMT1 plays a crucial but unknown role in the insect germline.

Dominant role of DNA methylation over H3K9me3 for IAP silencing in endoderm

Silencing of endogenous retroviruses (ERVs) is largely mediated by repressive chromatin modifications H3K9me3 and DNA methylation. On ERVs, these modifications are mainly deposited by the histone methyltransferase Setdb1 and by the maintenance DNA methyltransferase Dnmt1. Knock-out of either Setdb1 or Dnmt1 leads to ERV de-repression in various cell types. However, it is currently not known if H3K9me3 and DNA methylation depend on each other for ERV silencing. Here we show that conditional knock-out of Setdb1 in mouse embryonic endoderm results in ERV de-repression in visceral endoderm (VE) descendants and does not occur in definitive endoderm (DE). Deletion of Setdb1 in VE progenitors results in loss of H3K9me3 and reduced DNA methylation of Intracisternal A-particle (IAP) elements, consistent with up-regulation of this ERV family. In DE, loss of Setdb1 does not affect H3K9me3 nor DNA methylation, suggesting Setdb1-independent pathways for maintaining these modifications. Importantly, Dnmt1 knock-out results in IAP de-repression in both visceral and definitive endoderm cells, while H3K9me3 is unaltered. Thus, our data suggest a dominant role of DNA methylation over H3K9me3 for IAP silencing in endoderm cells. Our findings suggest that Setdb1-meditated H3K9me3 is not sufficient for IAP silencing, but rather critical for maintaining high DNA methylation.

Requirement of DNMT1 to orchestrate epigenomic reprogramming for NPM-ALK-driven lymphomagenesis.

Malignant transformation depends on genetic and epigenetic events that result in a burst of deregulated gene expression and chromatin changes. To dissect the sequence of events in this process, we used a T-cell-specific lymphoma model based on the human oncogenic nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) translocation. We find that transformation of T cells shifts thymic cell populations to an undifferentiated immunophenotype, which occurs only after a period of latency, accompanied by induction of the MYC-NOTCH1 axis and deregulation of key epigenetic enzymes. We discover aberrant DNA methylation patterns, overlapping with regulatory regions, plus a high degree of epigenetic heterogeneity between individual tumors. In addition, ALK-positive tumors show a loss of associated methylation patterns of neighboring CpG sites. Notably, deletion of the maintenance DNA methyltransferase DNMT1 completely abrogates lymphomagenesis in this model, despite oncogenic signaling through NPM-ALK, suggesting that faithful maintenance of tumor-specific methylation through DNMT1 is essential for sustained proliferation and tumorigenesis.

Characterization of Recombinant Maintenance DNA Methyltransferase (Dnmt1) and Application to DNA Methylation Analysis

A change doesn't occur to base sequence of DNA in the process of the human development. However, one fertilized egg differentiates into cells with sixty trillion of abundant individuality. The reason is DNA is methylated. The DNA methylated correctly is necessary for correct differentiation. Aberrant methylation induce various diseases including cancer, mental disorder and lifestyle disease.Elucidating essential of diseases needs analyzing methylation of DNA.Various DNA methylation analysis methods have been developed, among which bisulfite sequencing (BS-seq) is common.BS-seq has problems such as low sensitivity, which can not analyze cell types with insufficient sample amount, and may miss specific methylation of individual cells because it is an averaged data of cell population.Conventional BS-seq, hydroxymethylation (hmC), which is an intermediate of demethylation, is detected as methylation (mC). Since hmC is not recognized as mC in vivo, conventional BS-seq can not perform accurate DNA methylation analysis.New analytical methods are needed to assess pure DNA methylation even from small numbers of cells, such as single cells. In this study, we aim to develop a novel methylation analysis method that enables accurate methylation to be performed even with a small amount of DNA by repeating amplification and methylation of DNA using PCR and DNA (cytosine-5)-methyltransferase 1 (DNMT1).

Direct readout of heterochromatic H3K9me3 regulates DNMT1-mediated maintenance DNA methylation

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.

Enhanced processivity of Dnmt1 by monoubiquitinated histone H3.

DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.

The histone chaperone CAF-1 cooperates with the DNA methyltransferases to maintain Cd4 silencing in cytotoxic T cells.

The transcriptional repression of alternative lineage genes is critical for cell fate commitment. Mechanisms by which locus-specific gene silencing is initiated and heritably maintained during cell division are not clearly understood. To study the maintenance of silent gene states, we investigated how the Cd4 gene is stably repressed in CD8+ T cells. Through CRISPR and shRNA screening, we identified the histone chaperone CAF-1 as a critical component for Cd4 repression. We found that the large subunit of CAF-1, Chaf1a, requires the N-terminal KER domain to associate with the histone deacetylases HDAC1/2 and the histone demethylase LSD1, enzymes that also participate in Cd4 silencing. When CAF-1 was lacking, Cd4 derepression was markedly enhanced in the absence of the de novo DNA methyltransferase Dnmt3a but not the maintenance DNA methyltransferase Dnmt1. In contrast to Dnmt1, Dnmt3a deficiency did not significantly alter levels of DNA methylation at the Cd4 locus. Instead, Dnmt3a deficiency sensitized CD8+ T cells to Cd4 derepression mediated by compromised functions of histone-modifying factors, including the enzymes associated with CAF-1. Thus, we propose that the heritable silencing of the Cd4 gene in CD8+ T cells exploits cooperative functions among the DNA methyltransferases, CAF-1, and histone-modifying enzymes.

Stella safeguards the oocyte methylome by preventing de novo methylation mediated by DNMT1.

Postnatal growth of mammalian oocytes is accompanied by a progressive gain of DNA methylation, which is predominantly mediated by DNMT3A, a de novo DNA methyltransferase1,2. Unlike the genome of sperm and most somatic cells, the oocyte genome is hypomethylated in transcriptionally inert regions2-4. However, how such a unique feature of the oocyte methylome is determined and its contribution to the developmental competence of the early embryo remains largely unknown. Here we demonstrate the importance of Stella, a factor essential for female fertility5-7, in shaping the oocyte methylome in mice. Oocytes that lack Stella acquire excessive DNA methylation at the genome-wide level, including in the promoters of inactive genes. Such aberrant hypermethylation is partially inherited by two-cell-stage embryos and impairs zygotic genome activation. Mechanistically, the loss of Stella leads to ectopic nuclear accumulation of the DNA methylation regulator UHRF18,9, which results in the mislocalization of maintenance DNA methyltransferase DNMT1 in the nucleus. Genetic analysis confirmed the primary role of UHRF1 and DNMT1 in generating the aberrant DNA methylome in Stella-deficient oocytes. Stella therefore safeguards the unique oocyte epigenome by preventing aberrant de novo DNA methylation mediated by DNMT1 and UHRF1.

Critical Role of the UBL Domain in Stimulating the E3 Ubiquitin Ligase Activity of UHRF1 toward Chromatin

SummaryThe RING E3 ubiquitin ligase UHRF1 controls DNA methylation through its ability to target the maintenance DNA methyltransferase DNMT1 to newly replicated chromatin. DNMT1 recruitment relies on ubiquitylation of histone H3 by UHRF1; however, how UHRF1 deposits ubiquitin onto the histone is unknown. Here, we demonstrate that the ubiquitin-like domain (UBL) of UHRF1 is essential for RING-mediated H3 ubiquitylation. Using chemical crosslinking and mass spectrometry, biochemical assays, and recombinant chromatin substrates, we show that the UBL participates in structural rearrangements of UHRF1 upon binding to chromatin and the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1. Similar to ubiquitin, the UBL exerts its effects through a hydrophobic patch that contacts a regulatory surface on the “backside” of the E2 to stabilize the E2-E3-chromatin complex. Our analysis of the enzymatic mechanism of UHRF1 uncovers an unexpected function of the UBL domain and defines a new role for this domain in DNMT1-dependent inheritance of DNA methylation.

Chromatin structure and its chemical modifications regulate the ubiquitin ligase substrate selectivity of UHRF1

Mitotic inheritance of DNA methylation patterns is facilitated by UHRF1, a DNA- and histone-binding E3 ubiquitin ligase that helps recruit the maintenance DNA methyltransferase DNMT1 to replicating chromatin. The DNA methylation maintenance function of UHRF1 is dependent on its ability to bind chromatin, where it facilitates monoubiquitination of histone H3 at lysines 18 and 23, a docking site for DNMT1. Because of technical limitations, this model of UHRF1-dependent DNA methylation inheritance has been constructed largely based on genetics and biochemical observations querying methylated DNA oligonucleotides, synthetic histone peptides, and heterogeneous chromatin extracted from cells. Here, we construct semisynthetic mononucleosomes harboring defined histone and DNA modifications and perform rigorous analysis of UHRF1 binding and enzymatic activity with these reagents. We show that multivalent engagement of nucleosomal linker DNA and dimethylated lysine 9 on histone H3 directs UHRF1 ubiquitin ligase activity toward histone substrates. Notably, we reveal a molecular switch, stimulated by recognition of hemimethylated DNA, which redirects UHRF1 ubiquitin ligase activity away from histones in favor of robust autoubiquitination. Our studies support a noncompetitive model for UHRF1 and DNMT1 chromatin recruitment to replicating chromatin and define a role for hemimethylated linker DNA as a regulator of UHRF1 ubiquitin ligase substrate selectivity.

The effects of biological aging on global DNA methylation, histone modification, and epigenetic modifiers in the mouse germinal vesicle stage oocyte.

A cultural trend in developed countries is favoring a delay in maternal age at first childbirth. In mammals fertility and chronological age show an inverse correlation. Oocyte quality is a contributing factor to this multifactorial phenomenon that may be influenced by age-related changes in the oocyte epigenome. Based on previous reports, we hypothesized that advanced maternal age would lead to alterations in the oocyte’s epigenome. We tested our hypothesis by determining protein levels of various epigenetic modifications and modifiers in fully-grown (≥70 µm), germinal vesicle (GV) stage oocytes of young (10-13 weeks) and aged (69-70 weeks) mice. Our results demonstrate a significant increase in protein amounts of the maintenance DNA methyltransferase DNMT1 (P = 0.003) and a trend toward increased global DNA methylation (P = 0.09) with advanced age. MeCP2, a methyl DNA binding domain protein, recognizes methylated DNA and induces chromatin compaction and silencing. We hypothesized that chromatin associated MeCP2 would be increased similarly to DNA methylation in oocytes of aged female mice. However, we detected a significant decrease (P = 0.0013) in protein abundance of MeCP2 between GV stage oocytes from young and aged females. Histone posttranslational modifications can also alter chromatin conformation. Di-methylation of H3K9 (H3K9me2) is associated with permissive heterochromatin while acetylation of H4K5 (H4K5ac) is associated with euchromatin. Our results indicate a trend toward decreasing H3K9me2 (P = 0.077) with advanced female age and no significant differences in levels of H4K5ac. These data demonstrate that physiologic aging affects the mouse oocyte epigenome and provide a better understanding of the mechanisms underlying the decrease in oocyte quality and reproductive potential of aged females.

Epigenetic Enhancement of the Post-replicative DNA Mismatch Repair of Mammalian Genomes by a Hemi-mCpG-Np95-Dnmt1 Axis.

DNA methylation at C of CpG dyads (mCpG) in vertebrate genomes is essential for gene regulation, genome stability and development. We show in this study that proper functioning of post-replicative DNA mismatch repair (MMR) in mammalian cells relies on the presence of genomic mCpG, as well as on the maintenance DNA methyltransferase Dnmt1 independently of its catalytic activity. More importantly, high efficiency of mammalian MMR surveillance is achieved through a hemi-mCpG-Np95(Uhrf1)-Dnmt1 axis, in which the MMR surveillance complex(es) is recruited to post-replicative DNA by Dnmt1, requiring its interactions with MutSα, as well as with Np95 bound at the hemi-methylated CpG sites. Thus, efficiency of MMR surveillance over the mammalian genome in vivo is enhanced at the epigenetic level. This synergy endows vertebrate CpG methylation with a new biological significance and, consequently, an additional mechanism for the maintenance of vertebrate genome stability.

Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays.

The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

Profile of Dinshaw J. Patel

Small-noncoding RNA molecules are the dark matter of molecular biology. From small interfering (si) RNAs that silence invading pathogens to microRNAs that fine-tune gene expression, noncoding RNAs carry out a vast number of functions within the cell. “It was generally believed in the early days of molecular biology that RNA was just a passive intermediate on the pathway from DNA to protein, but noncoding RNA architecture and its interactions with partners is much more intricate and interesting,” says structural biologist Dinshaw J. Patel. Elected to the National Academy of Sciences in 2009, Patel has spent his career deciphering the shapes of biomolecules and principles underlying molecular complex formation. In his Inaugural Article, Patel, the Abby Rockefeller Mauze Chair in Experimental Therapeutics at the Memorial Sloan Kettering Cancer Center, explores how the PAZ-binding domain of PIWI proteins forms a pocket capable of binding the 3′-ends of piRNAs, noncoding RNA molecules that help maintain the integrity of germ-line DNA (1). Dinshaw J. Patel. Image courtesy of Memorial Sloan Kettering Cancer Center. Crystal structures of Thermus thermophilus Argonaute (labeled and color-coded by domains) with bound 5′-phosphorylated guide DNA (in red) as a binary complex ( A ) and with target RNA (in blue) as a ternary complex in B . Reprinted with permission from Macmillan Publishers Ltd., ref. 5. Crystal structure of mammalian maintenance DNA methyltransferase DNMT1 (containing color-coded CXXC, BAH1, BAH2, and MTase domains) bound to a 19-mer CG-containing DNA in the presence of cofactor AdoHcy. Reproduced from ref. 14. Patel was born in Mumbai, India in 1942 and grew up in the Zoroastrian community. His father was a civil engineer, and in an education-rich setting, Patel immersed himself in the sciences. “Because India was a developing country, science was what we learned in textbooks, with minimal practical experience,” Patel explains. …

The dynamics of DNA methylation and hydroxymethylation during amelogenesis.

Amelogenesis is a multistep process that relies on specific temporal and spatial signaling networks between the dental epithelium and mesenchymal tissues. Epigenetic modifications of key developmental genes in this process may be closely linked to a network of molecular events. However, the role of epigenetic regulation in amelogenesis remains unclear. Here, we have uncovered the spatial distributions of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) to determine epigenetic events in the mandibular incisors of mice. Immunohistochemistry and dot blotting showed that 5-hmC in ameloblasts increased from the secretory stage to the later maturation stage. We also demonstrated the distribution of 5-mC-positive ameloblasts with punctate nuclear labeling from sometime after the initiation of the secretory stage to the later maturation stage; however, dot blotting failed to detect this change. No obvious alteration of 5-mC/5-hmC staining in odontoblasts and dental pulp cells was observed. Concomitant with quantitative expression data, immunohistochemistry showed that maintenance DNA methyltransferase DNMT1 was highly expressed in immature dental epithelial cells and subsequently decreased at later stages of development. Meanwhile, de novo DNA methyltransferase Dnmt3a and Dnmt3b and DNA demethylase Tet family genes were universally expressed, except Tet1 that was highly expressed in immature dental epithelial cells. Thus, DNMT1 may sustain the undifferentiated status of dental epithelial cells through the maintenance of DNA methylation, while the hydroxylation of 5-mC may occur through the whole differentiation process by TET activity. Taken together, these data indicate that the dynamic changes of 5-mC and 5-hmC may be critical for the regulation of amelogenesis.

Biochemical characterization of maintenance DNA methyltransferase DNMT-1 from silkworm, Bombyx mori

DNA methylation is an important epigenetic mechanism involved in gene expression of vertebrates and invertebrates. In general, DNA methylation profile is established by de novo DNA methyltransferases (DNMT-3A, -3B) and maintainance DNA methyltransferase (DNMT-1). DNMT-1 has a strong substrate preference for hemimethylated DNA over the unmethylated one. Because the silkworm genome lacks an apparent homologue of de novo DNMT, it is still unclear that how silkworm chromosome establishes and maintains its DNA methylation profile. As the first step to unravel this enigma, we purified recombinant BmDNMT-1 using baculovirus expression system and characterized its DNA-binding and DNA methylation activity. We found that the BmDNMT-1 preferentially methylates hemimethylated DNA despite binding to both unmethylated and hemimethylated DNA. Interestingly, BmDNMT-1 formed a complex with DNA in the presence or absence of methyl group donor, S-Adenosylmethionine (AdoMet) and the AdoMet-dependent complex formation was facilitated by Zn2+ and Mn2+. Our results provide clear evidence that BmDNMT-1 retained the function as maintenance DNMT but its sensitivity to metal ions is different from mammalian DNMT-1.

The p53-induced lincRNA-p21 derails somatic cell reprogramming by sustaining H3K9me3 and CpG methylation at pluripotency gene promoters.

Recent studies have boosted our understanding of long noncoding RNAs (lncRNAs) in numerous biological processes, but few have examined their roles in somatic cell reprogramming. Through expression profiling and functional screening, we have identified that the large intergenic noncoding RNA p21 (lincRNA-p21) impairs reprogramming. Notably, lincRNA-p21 is induced by p53 but does not promote apoptosis or cell senescence in reprogramming. Instead, lincRNA-p21 associates with the H3K9 methyltransferase SETDB1 and the maintenance DNA methyltransferase DNMT1, which is facilitated by the RNA-binding protein HNRNPK. Consequently, lincRNA-p21 prevents reprogramming by sustaining H3K9me3 and/or CpG methylation at pluripotency gene promoters. Our results provide insight into the role of lncRNAs in reprogramming and establish a novel link between p53 and heterochromatin regulation.

LSD1 Influences Both Histone and DNA Methylation During Terminal Erythroid Maturation

The molecular mechanisms that drive the maturation of a committed erythroid progenitor to a functional red blood cell are incompletely understood. LSD1 (Lysine-Specific Histone Demethylase 1) is a widely expressed histone demethylase that plays an important role in erythroid maturation (Kereyni, elife, 2013). Although LSD1 is important for a number of biologic processes ranging from embryonic development to leukemogenesis, the molecular mechanisms underlying the influence of LSD1 on gene expression are incompletely understood. The goal of our study is to elucidate the molecular mechanisms by which LSD1 regulates erythroid gene expression and influences erythroid maturation. We hypothesize that LSD1 promotes specific patterns of histone and DNA methylation that facilitate gene expression changes necessary for normal erythroid maturation to occur. To address this hypothesis, the functional and molecular consequences of LSD1 knockdown were assessed in Extensively Self Renewing Erythroblasts (ESREs), a non-transformed, karyotypically normal model of terminal erythroid maturation (England, Blood, 2011). Primary fetal liver was cultured in the presence of EPO, SCF, IGF1 and dexamethasone to derive ESREs. The ESREs were capable of extensive ex-vivo expansion, doubling daily at the proerythroblast phase, however when matured, >90% of cells became benzidine positive and >65% enucleated within 3 days. Lentiviral-mediated shRNA was used to knock down LSD1 in expanding ESREs. Imaging flow cytometry done on maturation day 3 demonstrated that the knockdown cells had impairments in multiple facets of maturation, with larger cell and nuclear areas, higher kit expression, and lower rates of enucleation than the scramble control. LSD1 knockdown was also associated with impaired hemoglobin accumulation (78% vs. 95% benzidine positive; p<0.005). Treatment of ESREs with an inhibitor to LSD1 (Tranylcypromine; TCP) resulted in similar abnormalities in cell and nuclear size, kit expression, hemoglobin accumulation, and enucleation (40% vehicle vs.1% TCP). The functional deficits in maturation, including abnormal kit expression and low rates of enucleation, persisted on maturation day 4.To delineate the molecular mechanisms underlying this maturation impairment, RNA-seq was done in LSD1 knockdown and scramble control samples, and 230 differentially expressed genes (FDR<0.01) were identified using cuffdiff (Trapnell, Nat Biotech, 2013). Consistent with LSD1's role in erythroid maturation, Ingenuity Pathway Analysis identified multiple networks involving hemoglobin synthesis, and GATA1, EPO, and KLF1 were all predicted as upstream regulators (p-values of 8.24e10-11, 7.25 e10-6, and 3.86e10-4, respectively). To better understand how LSD1 influences gene expression, chromatin immunoprecipitation coupled with high throughput sequencing was used to identify sites of H3K4me2 binding in the differentially expressed genes. 214/230 differentially expressed genes were associated with sites of H3K4me2 occupancy. Quantitative ChIP demonstrated that LSD1 inhibition was associated with increases in H3K4me2 levels at a subset of these sites, however consistent with previous studies, global levels of H3K4me2, determined by Enzyme Linked Immunosorbent Assay (ELIZA), did not change significantly.Although it is known that LSD1 demethylates and stabilizes the maintenance DNA methyltransferase DNMT1 (Wang, Nat Genet 2009), the consequences of LSD1 loss on DNA methylation (5-methyl cytosine; 5-mC) have yet to be investigated. To gain a comprehensive understanding of how LSD1 regulates erythroid gene expression, changes in the level of 5-mC were assessed after knockdown or inhibition of LSD1. Global 5-mC levels, determined by ELIZA assay, were ∼30% lower in TCP treated samples than vehicle treated control (p<0.02) and western blot demonstrated a 3-fold decrease in DNMT1 protein in the TCP treated samples. Both methyl binding domain pull-down coupled with quantitative PCR and genome-wide bisulfite sequencing were utilized to assess changes in 5-mC levels in the differentially expressed genes. Loss of LSD1 was associated with significantly lower levels of 5-mC at several differentially expressed, erythroid-specific genes, such as bh1. Taken together, these data support the hypothesis that LSD1 influences both histone and DNA methylation at genes important for erythroid maturation. Disclosures:No relevant conflicts of interest to declare.