Structural Basis for MLL Cxxc Domain Protection against CpG DNA Methylation and the Essential Role of This Function in MLL-AF9 Leukemia.

Abstract Abstract 763 MLL is the target of chromosomal translocations which cause leukemias with poor prognosis. All leukemogenic MLL fusion proteins retain the CXXC domain which binds to nonmethylated CpG DNA. We present the solution structure of the MLL CXXC domain in complex with DNA, showing for the first time how the CXXC domain distinguishes nonmethylated from methylated CpG DNA. Based on the structure, we designed point mutations which disrupt DNA binding. Introduction of these mutations into MLL-AF9 results in increased DNA methylation of specific CpG nucleotides in Hoxa9, increased H3K9 methylation, decreased expression of Hoxa9 locus transcripts, loss of immortalization potential, and inability to induce leukemia in mice. These results establish that DNA binding by the CXXC domain and protection against DNA methylation is essential for MLL fusion leukemia. They also validate this interaction as a potential target for therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

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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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MBD2 acts as a repressor to maintain the homeostasis of the Th1 program in type 1 diabetes by regulating the STAT1-IFN-γ axis

Cell Death and Differentiation ◽

10.1038/s41418-021-00852-6 ◽

2021 ◽

Author(s):

Tiantian Yue ◽

Fei Sun ◽

Faxi Wang ◽

Chunliang Yang ◽

Jiahui Luo ◽

...

Keyword(s):

Dna Methylation ◽

Type 1 Diabetes ◽

Adoptive Transfer ◽

Nod Mice ◽

Transcriptional Level ◽

Clinical Settings ◽

Cpg Dna ◽

Ifn Γ

AbstractThe methyl-CpG-binding domain 2 (MBD2) interprets DNA methylome-encoded information through binding to the methylated CpG DNA, by which it regulates target gene expression at the transcriptional level. Although derailed DNA methylation has long been recognized to trigger or promote autoimmune responses in type 1 diabetes (T1D), the exact role of MBD2 in T1D pathogenesis, however, remains poorly defined. Herein, we generated an Mbd2 knockout model in the NOD background and found that Mbd2 deficiency exacerbated the development of spontaneous T1D in NOD mice. Adoptive transfer of Mbd2−/− CD4 T cells into NOD.scid mice further confirmed the observation. Mechanistically, Th1 stimulation rendered the Stat1 promoter to undergo a DNA methylation turnover featured by the changes of DNA methylation levels or patterns along with the induction of MBD2 expression, which then bound to the methylated CpG DNA within the Stat1 promoter, by which MBD2 maintains the homeostasis of Th1 program to prevent autoimmunity. As a result, ectopic MBD2 expression alleviated CD4 T cell diabetogenicity following their adoptive transfer into NOD.scid mice. Collectively, our data suggest that MBD2 could be a viable target to develop epigenetic-based therapeutics against T1D in clinical settings.

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Structural basis of DNA replication origin recognition by human Orc6 protein binding with DNA

Nucleic Acids Research ◽

10.1093/nar/gkaa751 ◽

2020 ◽

Vol 48 (19) ◽

pp. 11146-11161

Author(s):

Naining Xu ◽

Yingying You ◽

Changdong Liu ◽

Maxim Balasov ◽

Lee Tung Lun ◽

...

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Solution Structure ◽

Origin Recognition Complex ◽

Structural Basis ◽

Dna Replication Origin ◽

Dna Alterations ◽

Replication Initiator ◽

Binding Subunit ◽

Origin Recognition

Abstract The six-subunit origin recognition complex (ORC), a DNA replication initiator, defines the localization of the origins of replication in eukaryotes. The Orc6 subunit is the smallest and the least conserved among ORC subunits. It is required for DNA replication and essential for viability in all species. Orc6 in metazoans carries a structural homology with transcription factor TFIIB and can bind DNA on its own. Here, we report a solution structure of the full-length human Orc6 (HsOrc6) alone and in a complex with DNA. We further showed that human Orc6 is composed of three independent domains: N-terminal, middle and C-terminal (HsOrc6-N, HsOrc6-M and HsOrc6-C). We also identified a distinct DNA-binding domain of human Orc6, named as HsOrc6-DBD. The detailed analysis of the structure revealed novel amino acid clusters important for the interaction with DNA. Alterations of these amino acids abolish DNA-binding ability of Orc6 and result in reduced levels of DNA replication. We propose that Orc6 is a DNA-binding subunit of human/metazoan ORC and may play roles in targeting, positioning and assembling the functional ORC at the origins.

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Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

10.1101/2020.08.11.243204 ◽

2020 ◽

Cited By ~ 1

Author(s):

Tilak Kumar Gupta ◽

Sven Klumpe ◽

Karin Gries ◽

Steffen Heinz ◽

Wojciech Wietrzynski ◽

...

Keyword(s):

Membrane Integrity ◽

Point Mutations ◽

Binding Pocket ◽

Thylakoid Membranes ◽

Lipid Binding ◽

Structural Basis ◽

Amphipathic Helices ◽

Cryo Electron Microscopy

AbstractVesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the ring’s lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress.

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Role of DNA methylation and methyl-DNA binding proteins in the repression of 5-lipoxygenase promoter activity

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids ◽

10.1016/j.bbalip.2009.09.003 ◽

2010 ◽

Vol 1801 (1) ◽

pp. 49-57 ◽

Cited By ~ 18

Author(s):

Careen Katryniok ◽

Nicole Schnur ◽

Ad Gillis ◽

Andreas von Knethen ◽

Bernd L. Sorg ◽

...

Keyword(s):

Dna Methylation ◽

Dna Binding ◽

Promoter Activity ◽

Binding Proteins ◽

Dna Binding Proteins

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Autoantigenic Targets of B-Cell Receptors (BCR) Derived From Chronic Lymphocytic Leukemias Bind to and Induce Proliferation of Leukemic Cells.

Blood ◽

10.1182/blood.v120.21.2884.2884 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2884-2884

Author(s):

Klaus-Dieter Preuss ◽

Gerhard Held ◽

Natalie Fadle ◽

Evi Regitz ◽

Maria Kemele ◽

...

Keyword(s):

B Cell ◽

Conflicts Of Interest ◽

Specific Binding ◽

Cell Receptor ◽

Convincing Evidence ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Long Time ◽

First Time

Abstract Abstract 2884 Background Auto-antigenic targets of the B-cell receptor (BCR) derived from malignant cells in chronic lymphocytic leukemia (CLL) might play a role in the pathogenesis of this neoplasm. Patients and Methods In order to identify autoantigenic targets of CLL-derived BCR we screened human tissue-derived protein macroarrays with Fab fragments obtained by papain treatment of CLL cells derived from 50 consecutive cases. Antigens were biochemically and molecularly characterized and recombinantly expressed. Results An autoantigenic target was identified for 12/50 (24%) of the cases, with 3 autoantigens being the target of the BCR from two patients each. CLL-BCR derived from the same stereotype subset recognized the same antigen, but differed epitopes. By flow cytometry using flag-tagged recombinantly expressed autoantigens binding of antigen to the surface of CLL was demonstrated, which was specific for the CLL cells from which the BCR used for the identification of the respective autoantigen was derived. Moreover, binding of the autoantigen to the respective leukemic cells induced specific activation as shown by increased cytoplasmic calcium concentration, induced MYC expression and proliferation of leukemic CLL cells as demonstrated by a proliferation assay (EZ4U). Conclusions Autoantigens are frequent targets of CLL-derived BCR. Their specific binding to and induction of proliferation in respective leukemic cells, which has been demonstrated for the first time, provide the most convincing evidence to date for the long-time hypothesized role of autoantigens in the pathogenesis of chronic lymphocyte leukemia. Supported by Sander-Stiftung (Munich, Germany) Disclosures: No relevant conflicts of interest to declare.

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The structural basis of DNA binding by the single-stranded DNA-binding protein from Sulfolobus solfataricus

Biochemical Journal ◽

10.1042/bj20141140 ◽

2015 ◽

Vol 465 (2) ◽

pp. 337-346 ◽

Cited By ~ 19

Author(s):

Roland Gamsjaeger ◽

Ruvini Kariawasam ◽

Adrian X. Gimenez ◽

Christine Touma ◽

Elysse McIlwain ◽

...

Keyword(s):

Dna Binding ◽

Solution Structure ◽

Binding Protein ◽

Sulfolobus Solfataricus ◽

Dna Binding Protein ◽

Structural Basis ◽

Single Stranded Dna

We present the 3D solution structure of the canonical SSB from the crenarchaeote Sulfolobus solfataricus bound to single-stranded DNA and compare this structure with human homologues.

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New insertions-induced Hb H disease

Genetics & Applications ◽

10.31383/ga.vol5iss1pp64-67 ◽

2021 ◽

Vol 5 (1) ◽

pp. 64

Author(s):

Marzieh Alipour ◽

Sara Afzali ◽

Khalil Khashei

Keyword(s):

Southeast Asia ◽

Rare Variants ◽

Point Mutations ◽

Clinical Importance ◽

High Impact ◽

New Findings ◽

High Incidence ◽

Hb H Disease ◽

First Time

In spite of high incidence of α-thalassemia as the most common inherited disorder of hemoglobin (Hb) production in Southeast Asia, the role of point mutations in this aspect not still well known. This fact can lead to missing rare variants of α-thalassemia mutations by the routine screening, which they may need to be screened for possibility of causing Hb H disease. In this study we found two insertions in alpha1 gene which cause to Hb H disease. One of the insertions, 108/109, is a new findings and another one, codon 44, is the mutation which has been followed for the first time. These new molecular findings about changes in α-globin production which results in decreased of hemoglobin (Hb) value, have high-impact clinical importance.

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Glanzmann Mutations : New Allelic Variants and Role of the β3 Lys253 in the αIIb/β3 Interaction Highlighted by the Lys253Met Substitution.

Blood ◽

10.1182/blood.v114.22.1312.1312 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1312-1312

Author(s):

Vincent Jallu ◽

Alexandre De Brevern ◽

Simon Panzer ◽

Marie-Francoise Torchet ◽

Cecile Kaplan-Gouet

Keyword(s):

Rna Processing ◽

Conflicts Of Interest ◽

Single Point ◽

Point Mutations ◽

Single Point Mutation ◽

Platelet Surface ◽

Cos Cells ◽

Single Point Mutations ◽

Complex Expression

Abstract Abstract 1312 Poster Board I-336 Introduction Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation. GT results from defects of the platelet fibrinogen receptor αIIbβ3. GT mutations provide useful tools for structure-function relationship studies of αIIbβ3. Patient and methods Genomic DNA from 6 patients has been amplified for αIIb and β3 promoters and exons sequences. PCR products were directly sequenced. Potential RNA processing alterations have been studied in silico by using Genscan, NNSPLICE and ESEFinder online tools. When no RNA splicing anomaly was predicted, the effect of single point mutation on αIIbβ3 expression has been studied by using transiently transfected Cos cells. Finally, structural consequences of amino acid substitutions has been studied using the published model of αIIbβ3 (code PDB 2VDL) and structural modelling. Results 7 new mutations have been characterized. 1 deletion / insertion, 2 single point mutations inducing stop codon and 1 resulting in splicing site disruption were identified. The 3 last identified single point mutations were not predicted to affect normal RNA processing but has been shown to prevent normal expression of mutant αIIbβ3 at the surface of Cos cells. The p.Meth118Arg and p.Gly221Asp substitutions that induce both important steric hindrance and charge modifications, are located inside the β-I domain of β3. So they should deeply alter the proper folding of the β-I domain, preventing the complex expression at the platelet surface. On the other hand, the p.Lys253Met protrudes from the β-I domain toward the αIIb β-propeller. A structural model of the Met253 β-I mutant has been done. An estimation of the direct electrostatic and desolvation free energies of interaction between the β-I domain surface and the αIIb β-propeller indicated that rather than the presence of a methionine, it is the lost of the Lys253 which is responsible for the complex expression defect. Conclusion Seven new GT mutations have been identified and the p.Lys253Met substitution helped to define a key role of the Lys253 in the αIIb β-propeller / β3 β-I domains interaction. Disclosures No relevant conflicts of interest to declare.

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The SQSTM1-NUP214 Fusion Protein Cooperates with Crm1 to Activate Hoxa Genes and Drives Leukemogenesis in Mice

Blood ◽

10.1182/blood-2019-130530 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1434-1434

Author(s):

Waitman K. Aumann ◽

Sei-Gyung K. Sze ◽

Veerain Gupta ◽

Katelyn Ripple ◽

Sarah Port ◽

...

Keyword(s):

Fusion Protein ◽

Chromatin Immunoprecipitation ◽

Nuclear Export ◽

Conflicts Of Interest ◽

Point Mutations ◽

Heterologous Proteins ◽

Hematopoietic Progenitors ◽

Fusion Methods ◽

Nuclear Export Receptor

Background: The NUP98 and NUP214 nucleoporins (NUPs) are recurrently fused to heterologous proteins in leukemia. The resulting chimeric oncoproteins retain the NUP phenylalanine-glycine (FG) repeat motifs that mediate interaction with the nuclear export receptor Crm1. NUP fusion leukemias are characterized by HOXA gene upregulation; however, their molecular pathogenesis remains poorly understood. To investigate the role of Crm1 in mediating the leukemogenic properties of NUP chimeric proteins, we studied the SQSTM1-NUP214 fusion. Methods: We synthesized a SQSTM1-NUP214 fusion protein which retains only a short C-terminal portion of NUP214 containing FG motifs that mediate interaction with Crm1, and then introduced point mutations targeting these FG motifs (SQSTM1-NUP214FGmut). We compared the activity of these two fusion proteins using co-immunoprecipitation with CRM1, methylcellulose colony assays, murine transplantation, RT-qPCR, and chromatin immunoprecipitation. Results: We found that the ability of the SQSTM1-NUP214FGmut protein to interact with Crm1 was reduced by more than 50% compared with SQSTM1-NUP214. Further, mutation of FG motifs affected transforming potential: while SQSTM1-NUP214 conferred robust colony formation to transduced hematopoietic progenitors in a serial replating assay, the effect of SQSTM1-NUP214FGmut was greatly diminished. Moreover, SQSTM1-NUP214 caused myeloid leukemia in all transplanted mice (6/6), whereas none of the SQSTM1-NUP214FGmut reconstituted mice developed leukemia (0/7). These oncogenic effects coincided with the ability of SQSTM1-NUP214 and SQSTM1-NUP214FGmut to upregulate the expression of Hoxa and Meis1 genes in hematopoietic progenitors. Indeed, chromatin immunoprecipitation assays demonstrated that impairing the interaction of SQSTM1-NUP214 with Crm1 reduced binding of the fusion protein to Hoxa and Meis1 loci. Conclusions: These findings highlight the importance of Crm1 in mediating the leukemogenic properties of SQSTM1-NUP214, and suggest a conserved role of Crm1 in recruiting oncoproteins to their effector genes. Disclosures No relevant conflicts of interest to declare.

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