Structure study of UHRF1, recoginition of epigenetic marks.

Two major epigenetic traits, histone modifications and DNA methylation, regulate various chromatin-template processes in mammals. The pattern of these epigenetic traits is cooperatively established in early embryogenesis and cell development, and inherited during the cell cycle. UHRF1 (also known as Np95 or ICBP90) is believed to play an important role in linking the two major epigenetic traits. UHRF1 has five functional domains, UBL, Tandem Tudor (TTD), pland homeo domain (PHD), SET and RING-associated doain (SRA) and RING finger. To maintain DNA methylation pattern, UHRF1 recognizes hemi-methylated DNA generated during DNA replication through interactions with its SRA domain, and recruit maintenance of DNA methyltransferase Dnmt1 to the site [1], [2]. UHRF1 also recognizes histone H3 containing tri-methylated Lys9 (H3K9me3) via its TTD-PHD moiety. [3]. To obtain the structural basis for recognition of epigenetic marks by UHRF1, we determined the crystal structure of the SRA domain in complex with hemi-methylated DNA. The structure showed that the DNA binding caused a loop and an N-terminal tail of the SRA domain. Interestingly, the methyl-cytosine base at the hemi-methylation site was flipped out from the DNA helix, which has not observed in other DNA binding proteins. These results suggest that the Base flip out mechanism is important event for maintenance of DNA methylation. We also determined the crystal structure of TTD-PHD region of UHRF1 in complex with H3K9me3 peptide. To our surprise, the linker region between the reader modules, which is predicted as an intrinsically disorder, was formed a stable structure with binding to the groove of TTD and plays an essential role in the formation of histone H3 binding hole between the reader modules. The structure revealed how multiple histone modifications were simultaneously decoded by the linked histone reader modules of UHRF1.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2009316117 ◽

2020 ◽

Vol 117 (31) ◽

pp. 18439-18447

Author(s):

Wendan Ren ◽

Huitao Fan ◽

Sara A. Grimm ◽

Yiran Guo ◽

Jae Jin Kim ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Histone Modifications ◽

Dna Methyltransferase ◽

Histone H3 ◽

Genomic Stability ◽

Epigenetic Modifications ◽

Global Dna Methylation ◽

Replication Foci ◽

Direct Readout

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.

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

10.1101/2020.04.27.064493 ◽

2020 ◽

Author(s):

Wendan Ren ◽

Huitao Fan ◽

Sara A Grimm ◽

Yiran Guo ◽

Jae Jin Kim ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Histone Modifications ◽

Dna Methyltransferase ◽

Histone H3 ◽

Genomic Stability ◽

Epigenetic Modifications ◽

Global Dna Methylation ◽

Replication Foci ◽

Direct Readout

ABSTRACTIn 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.

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Faculty Opinions recommendation of Interplay between two epigenetic marks. DNA methylation and histone H3 lysine 9 methylation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1009101.118757 ◽

2002 ◽

Author(s):

Eric Richards

Keyword(s):

Dna Methylation ◽

Histone H3 ◽

Epigenetic Marks

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Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

Scientific Reports ◽

10.1038/s41598-021-94528-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Krystyna Ślaska-Kiss ◽

Nikolett Zsibrita ◽

Mihály Koncz ◽

Pál Albert ◽

Ákos Csábrádi ◽

...

Keyword(s):

Dna Methylation ◽

Dna Binding ◽

Binding Affinity ◽

Dna Methyltransferase ◽

Restriction Enzymes ◽

Dna Binding Protein ◽

E Coli ◽

Dna Binding Affinity ◽

Higher Eukaryotes

AbstractTargeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

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DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation

Nature Communications ◽

10.1038/s41467-021-22665-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Wendan Ren ◽

Huitao Fan ◽

Sara A. Grimm ◽

Jae Jin Kim ◽

Linhui Li ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Histone H3 ◽

Genomic Stability ◽

Mammalian Genome ◽

Dna Methyltransferase 1 ◽

Chromatin Modifications ◽

Direct Link ◽

Histone H4 ◽

Radiation Induced

AbstractDNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically ‘recognizes’ H4K20me3 via its first bromo-adjacent-homology domain (DNMT1BAH1). Engagement of DNMT1BAH1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1’s activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability.

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Thymoquinone Is a Multitarget Single Epidrug That Inhibits the UHRF1 Protein Complex

Genes ◽

10.3390/genes12050622 ◽

2021 ◽

Vol 12 (5) ◽

pp. 622

Author(s):

Omeima Abdullah ◽

Ziad Omran ◽

Salman Hosawi ◽

Ali Hamiche ◽

Christian Bronner ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Ring Finger ◽

Macromolecular Complex ◽

Anticancer Activities ◽

Histone Deacetylase 1 ◽

Promoter Dna Methylation ◽

New Gene ◽

Regulatory Effects ◽

Promoter Dna

Silencing of tumor suppressor genes (TSGs) through epigenetic mechanisms, mainly via abnormal promoter DNA methylation, is considered a main mechanism of tumorigenesis. The abnormal DNA methylation profiles are transmitted from the cancer mother cell to the daughter cells through the involvement of a macromolecular complex in which the ubiquitin-like containing plant homeodomain (PHD), and an interesting new gene (RING) finger domains 1 (UHRF1), play the role of conductor. Indeed, UHRF1 interacts with epigenetic writers, such as DNA methyltransferase 1 (DNMT1), histone methyltransferase G9a, erasers like histone deacetylase 1 (HDAC1), and functions as a hub protein. Thus, targeting UHRF1 and/or its partners is a promising strategy for epigenetic cancer therapy. The natural compound thymoquinone (TQ) exhibits anticancer activities by targeting several cellular signaling pathways, including those involving UHRF1. In this review, we highlight TQ as a potential multitarget single epidrug that functions by targeting the UHRF1/DNMT1/HDAC1/G9a complex. We also speculate on the possibility that TQ might specifically target UHRF1, with subsequent regulatory effects on other partners.

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The major histocompatibility complex class II promoter-binding protein RFX (NF-X) is a methylated DNA-binding protein.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6810 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6810-6818 ◽

Cited By ~ 33

Author(s):

X Y Zhang ◽

N Jabrane-Ferrat ◽

C K Asiedu ◽

S Samac ◽

B M Peterlin ◽

...

Keyword(s):

Dna Methylation ◽

Dna Binding ◽

Dna Sequences ◽

Protein Complexes ◽

Binding Protein ◽

Dna Binding Protein ◽

Class Ii ◽

Major Histocompatibility ◽

Methylated Dna ◽

Mammalian Protein

A mammalian protein called RFX or NF-X binds to the X box (or X1 box) in the promoters of a number of major histocompatibility (MHC) class II genes. In this study, RFX was shown to have the same DNA-binding specificity as methylated DNA-binding protein (MDBP), and its own cDNA was found to contain a binding site for MDBP in the leader region. MDBP is a ubiquitous mammalian protein that binds to certain DNA sequences preferentially when they are CpG methylated and to other related sequences, like the X box, irrespective of DNA methylation. MDBP from HeLa and Raji cells formed DNA-protein complexes with X-box oligonucleotides that coelectrophoresed with those containing standard MDBP sites. Furthermore, MDBP and X-box oligonucleotides cross-competed for the formation of these DNA-protein complexes. DNA-protein complexes obtained with MDBP sites displayed the same partial supershifting with an antiserum directed to the N terminus of RFX seen for complexes containing an X-box oligonucleotide. Also, the in vitro-transcribed-translated product of a recombinant RFX cDNA bound specifically to MDBP ligands and displayed the DNA methylation-dependent binding of MDBP. RFX therefore contains MDBP activity and thereby also EF-C, EP, and MIF activities that are indistinguishable from MDBP and that bind to methylation-independent sites in the transcriptional enhancers of polyomavirus and hepatitis B virus and to an intron of c-myc.

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Structural insights into CpG-specific DNA methylation by human DNA methyltransferase 3B

Nucleic Acids Research ◽

10.1093/nar/gkaa111 ◽

2020 ◽

Vol 48 (7) ◽

pp. 3949-3961 ◽

Cited By ~ 5

Author(s):

Chien-Chu Lin ◽

Yi-Ping Chen ◽

Wei-Zen Yang ◽

James C K Shen ◽

Hanna S Yuan

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Cytosine Methylation ◽

Catalytic Domain ◽

De Novo ◽

Water Channel ◽

Dna Methyltransferases ◽

Structural Basis ◽

Cpg Sites ◽

Methylation Patterns

Abstract DNA methyltransferases are primary enzymes for cytosine methylation at CpG sites of epigenetic gene regulation in mammals. De novo methyltransferases DNMT3A and DNMT3B create DNA methylation patterns during development, but how they differentially implement genomic DNA methylation patterns is poorly understood. Here, we report crystal structures of the catalytic domain of human DNMT3B–3L complex, noncovalently bound with and without DNA of different sequences. Human DNMT3B uses two flexible loops to enclose DNA and employs its catalytic loop to flip out the cytosine base. As opposed to DNMT3A, DNMT3B specifically recognizes DNA with CpGpG sites via residues Asn779 and Lys777 in its more stable and well-ordered target recognition domain loop to facilitate processive methylation of tandemly repeated CpG sites. We also identify a proton wire water channel for the final deprotonation step, revealing the complete working mechanism for cytosine methylation by DNMT3B and providing the structural basis for DNMT3B mutation-induced hypomethylation in immunodeficiency, centromere instability and facial anomalies syndrome.

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Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

Cellular Physiology and Biochemistry ◽

10.1159/000494598 ◽

2018 ◽

Vol 50 (4) ◽

pp. 1376-1397 ◽

Cited By ~ 5

Author(s):

Yanhui Zhai ◽

Zhiren Zhang ◽

Hao Yu ◽

Li Su ◽

Gang Yao ◽

...

Keyword(s):

Dna Methylation ◽

Somatic Cell ◽

Histone Modifications ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Histone H3 ◽

Dna Demethylation ◽

Epigenetic Reprogramming ◽

Expression Levels ◽

Fetal Fibroblasts

Background/Aims: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells’ affects SCNT embryos development and the crosstalk between epigenetic signals. Results: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.

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