rabbit anti morc2  (Bethyl)

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: MORC2 preferentially associates with dsDNA via a C-terminal region. ( A ) Domain architecture of human MORC2. Structured domains are colored green. Coiled coil domains (CC), S5 transducer domain of the GHKL ATPase module (S5), a CW-type zinc finger domain (CW) and a predicted chromodomain (CD) are indicated. Purified MORC2 was cross-linked to DNA with 1 mM mechlorethamine for 30 min at 37°C or cross-linked to DNA by UV light at 254 nm for 10 min on ice, and samples were digested by trypsin. Resulting fragments were enriched by TiO 2 and analyzed by mass spectrometry (‘Materials and methods’ section). Identified amino acid positions cross-linked to DNA are denoted by a black cross. Positively charged residues and phosphorylation sites mutated in this study are noted in pink and orange, respectively, beneath the primary structure. ( B ) SDS-PAGE gel of purified human MORC2 protein (3 μg) stained with Coomassie blue stain. Contaminant band marked with an asterisk . ( C ) MORC2 DNA binding to DNA sequences of different AT/GC content as assessed by fluorescence anisotropy. Dephosphorylated MORC2 was titrated and incubated with 1 nM 5′-FAM-labeled 35 base pair duplex DNA with a high GC (71% GC content), high AT (31% GC content) or a random sequence with 49% GC content (‘Materials and methods’ section). Binding curves were fit with a single site quadratic binding equation. Error bars correspond to the standard deviation between three replicate experiments. ( D ) MORC2 binding to a 500 bp DNA substrate assessed by gel shift. Increasing concentrations of dephosphorylated MORC2 was incubated with 20 nM of duplex DNA and resolved on a 3–12% gradient Native PAGE gel (‘Materials and methods’ section). ( E ) MORC2 binding to a 1000 bp DNA substrate assessed by gel shift. Increasing concentrations of dephosphorylated MORC2 was incubated with 20 nM of duplex DNA and resolved on a 3–12% gradient Native PAGE gel (‘Materials and methods’ section). ( F ) MORC2 nucleic acid binding as measured by fluorescence anisotropy. Dephosphorylated MORC2 was titrated and incubated with 1 nM 5′-FAM-labeled 149 bp Widom 601 dsDNA, nucleosome, cruciform DNA, 35 nucleotide ssRNA and 35 base pair ssDNA (‘Materials and methods’ section). Binding curves were fit as in 1C except for the cruciform DNA which was fit with a Hill equation. Error bars correspond to the standard deviation between three replicate experiments. ( G ) MORC2 association with DNAs of varying topologies. Dephosphorylated MORC2 (150 nM) was incubated with 5′-FAM-labeled 35 base pair duplex DNA (1 nM), and positively supercoiled, negatively supercoiled, or relaxed plasmid DNA was titrated into the reactions (‘Materials and methods’ section). Binding curves were fit using an inhibition curve with a variable response. Error bars correspond to the standard deviation between three replicate experiments. ( H ) Assessment of DNA binding by dephosphorylated wild-type, aspartate mutant and 1–603 MORC2. MORC2 constructs were titrated and incubated with a FAM-labeled 35 base pair duplex DNA (1 nM) (‘Materials and methods’ section). Binding curves were fit as in 1C. Error bars correspond to the standard deviation between three replicate experiments.

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Purification, Mass Spectrometry, Phospho-proteomics, SDS Page, Staining, Binding Assay, Fluorescence, Incubation, Labeling, Sequencing, Standard Deviation, Gel Shift, Clear Native PAGE, Plasmid Preparation, Inhibition, Mutagenesis, Construct

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: Guide RNA sequences used to generate MORC2 knockout cells

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Knock-Out

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: PCR primers used to verify MORC2 knockout cells

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Knock-Out

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: MORC2 phosphorylation influences DNA binding and nuclear localization. ( A ) Phosphorylation reduces MORC2 affinity for DNA. Dephosphorylated, phosphorylated, phosphodead and phosphomimetic MORC2 were assessed for DNA binding using fluorescence anisotropy. MORC2 was titrated with FAM-labeled 35 base pair duplex DNA (1 nM) (‘Materials and methods’ section). Binding curves were fit using a quadratic binding equation. Error bars correspond to the standard deviation between three replicate experiments. ( B ) Effect of phosphorylation mutations on MORC2 localization in interphase HeLa cells. Representative confocal microscopy images of interphase HeLa cells overexpressing EGFP-wild-type MORC2, EGFP-MORC2 1–603, EGFP- MORC2 734–771, EGFP- phosphodead MORC2 and EGFP- phosphomimetic MORC2.

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Phospho-proteomics, Binding Assay, Fluorescence, Labeling, Standard Deviation, Confocal Microscopy

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: DNA binding by MORC2 reduces ATPase activity. ( A ) Assessment of MORC2 ATPase activity. Dephosphorylated wild-type, 1–603, DNA binding deficient aspartate mutant, and ATP hydrolysis deficient mutant E35A, and ATP binding deficient mutant N39A MORC2 (1 μM) were incubated with 1 mM ATP for 45 min at 37°C either in the presence or absence of a 35 base pair DNA (2 μM). Inorganic phosphate released was quantified by malachite green (‘Materials and methods’ section). Error bars correspond to the standard deviation between three replicate experiments. ( B ) MORC2 ATPase activity is reduced in the presence of DNA. Dephosphorylated MORC2 (1 μM) was incubated with 1 mM ATP for 45 min at 37°C with a titration of a 35 base pair duplex DNA. Inorganic phosphate released was quantified by malachite green (‘Materials and methods’ section). Data were fit to an inhibition curve. Error bars correspond to the standard deviation between three replicate experiments. ( C ) Michaelis–Menten analysis of MORC2 ATPase activity in the presence of DNA. Dephosphorylated MORC2 (1 μM) was incubated with an ATP titration for 45 min at 37°C in the presence of various concentrations of 35mer DNA (0–1 μM). Inorganic phosphate released was quantified by malachite green (‘Materials and methods’ section). Data were fit to a Michaelis–Menten model of enzyme kinetics. Error bars correspond to the standard deviation between three replicate experiments.

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Binding Assay, Activity Assay, Mutagenesis, Incubation, Standard Deviation, Titration, Inhibition

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: MORC2 homodimerizes via two distinct interfaces. ( A ) Dephosphorylated full-length MORC2 dimerizes in the presence and absence of AMP-PNP. Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) experiments with wild-type (monomer MW = 117 kDa) and 1–603 (monomer MW = 70 kDa) MORC2 in the presence or absence of 1 mM AMP-PNP. 2 mg/ml of MORC2 was applied to a WC-030 column (Wyatt Technology), and elution was monitored by absorption at 280 nm (left y -axis). MALS analysis of molecular weight is shown on the right y -axis, and average molecular weight calculations are shown across the center of each peak. ( B ) AlphaFold Multimer model of coiled coil 3 domain dimer. Chain A is colored gray and chain B is colored by pLDDT score. ( C ) MORC2 coiled coil 3 dimerizes in solution. SEC-MALS experiment performed with 1.5 mg/ml of purified MORC2 coiled coil 3 (monomer MW = 16 kDa) on a WC-010 column (Wyatt Technology). Elution was monitored by absorption at 280 nm (left y -axis). MALS analysis of molecular weight is shown on the right y -axis, and the average molecular calculation is shown across the center of the peak.

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Size-exclusion Chromatography, Multi-Angle Light Scattering, Molecular Weight, Purification

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: MORC2 can capture circular DNA substrates. ( A ) Schematic of assay to assess MORC2 capture of linear or circular DNA substrates. ( B ) Dephosphorylated, N-terminally-MBP-tagged wild-type, aspartate mutant and E35A MORC2 (600 nM) were incubated with supercoiled or linear pUC19 (100 nM). Samples were then added to amylose resin and washed in either low salt (50 mM NaCl) or high salt (400 mM NaCl) containing buffers before eluting the samples from the beads with maltose. Eluted samples were treated with proteinase K. DNA was resolved on a 1% (w/v) TAE agarose gel. Quantification of the band intensity from input and retained DNA bands normalized to the background are shown below each gel. Error bars correspond to the standard deviation between three replicate experiments. ( C ) Schematic of assay to assess MORC2 association with two circular DNA substrates. ( D ) A biotin-tagged DNA was conjugated to streptavidin magnetic beads to create a pseudo circular substrate. Dephosphorylated MORC2 (600 nM) was incubated with 20 μl of the beads in the presence or absence of 1 mM AMP-PNP. Supercoiled pBlueScript plasmid DNA (200 nM) was added, and 1 mM AMP-PNP was added or omitted before washing the beads with either low salt (50 mM NaCl) or high salt (400 mM NaCl) containing buffer. Samples were resuspended in 1X CutSmart buffer (New England Biolabs). DNA was released from the beads by digestion with ScaI and SbfI at 37°C for 1 h before proteinase K treatment. DNA was resolved on a 1% (w/v) TAE agarose gel. The intensity of the first DNA and second DNA substrate bands were quantified, normalized to the background and are presented as a ratio of second DNA:first DNA band intensity. Error bars correspond to the standard deviation between three replicate experiments. ( E ) Schematic of assay to assess MORC2 association with three circular DNA substrates. A biotin-tagged DNA was conjugated to streptavidin magnetic beads to create a pseudo-circular substrate. Dephosphorylated MORC2 (600 nM) was incubated with 20 μl of the beads. Supercoiled pBlueScript plasmid DNA (100 nM) and pBlueScript-601 plasmid DNA (100 nM) were added and 1 mM AMP-PNP was added or omitted before washing the beads with either low salt (50 mM NaCl) or high salt (400 mM NaCl) containing buffer. Samples were resuspended in 1X CutSmart buffer (New England Biolabs). DNA was released from the beads by digestion with ScaI and SbfI at 37°C for 1 h before proteinase K treatment. DNA was resolved on a 1% (w/v) TAE agarose gel.

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Mutagenesis, Incubation, Agarose Gel Electrophoresis, Standard Deviation, Magnetic Beads, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: DNA binding regulates MORC2 gene silencing activity in cells. ( A ) Schematic of RNAseq experiment. MORC2 constructs were added to HeLa cells lacking MORC2. MORC2 expression was induced for 48 h with doxycycline after which RNAs were extracted and sequenced. Volcano plots of RNAseq reads for overexpression of wild-type MORC2 versus control, overexpression of aspartate mutant MORC2 versus control, and wild-type MORC2 versus aspartate mutant MORC2 with spike normalization (‘Materials and methods’ section). Significant upregulated genes are shown in green, and significant downregulated genes are shown in purple from three biological replicates. Significant genes are classified as those that meet the fold change > 1.5 and FDR > 0.05 cutoffs. ( B ) Fold repression (-log 2 fold change) analysis of intronless and long-exon containing genes exhibiting a significant degree of repression after overexpression of wild-type MORC2 ( n = 75) in comparison to their fold repression after overexpression of aspartate mutant MORC2. Error bars correspond to the minimum and maximum values and the central bar represents the median value. Dotted lines connect genes between the two conditions. ( C ) Average fold repression (-log 2 fold change) of retrotransposon subfamilies as measured by RNAseq in cells with overexpression of wild-type or aspartate mutant MORC2 versus control cells using the TEtranscripts tool. Error bars represent the standard error between the elements in each subfamily (L1Hs n = 1, L1PA n = 30, L1M n = 86 and LTR-retrotransposons n = 347). ( D ) Model of how MORC2 engages DNA to promote compaction. MORC2 contains a DNA binding region between the GHKL domain and coiled coil 3 domain dimerization interfaces. DNA binding inside the lumen of the dimer communicates to the ATPase domain, to favor an ATP-bound homodimer conformation that is incompatible with ATP hydrolysis. MORC2 association with multiple DNA segments may allow MORC2 to bridge distal regions of DNA to contribute to compaction.

Article Snippet: Beads were resuspended in 1X TBS (1 ml), with the addition of 30 μg either MORC2 antibody (Bethyl Lab #A300-149A), GFP antibody (Invitrogen #MA5-15256) or Rabbit IgG Isotype Control (Invitrogen #02–6102), and the mixture was incubated at 4°C with end-over-end mixing on a Tube Revolver (Fisher Scientific cat. no. 88861051) for 16–24 h. To immunoprecipitate MORC2, different fractions of cell lysates were incubated with Rabbit-IgG Isotype Control antibody conjugated beads at 4°C for 1 h. Lysate was clarified by centrifugation (3220 × g , 30 min, 4°C) and transferred to a new tube.

Techniques: Binding Assay, Activity Assay, Construct, Expressing, Over Expression, Control, Mutagenesis, Comparison

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: MORC2 preferentially associates with dsDNA via a C-terminal region. ( A ) Domain architecture of human MORC2. Structured domains are colored green. Coiled coil domains (CC), S5 transducer domain of the GHKL ATPase module (S5), a CW-type zinc finger domain (CW) and a predicted chromodomain (CD) are indicated. Purified MORC2 was cross-linked to DNA with 1 mM mechlorethamine for 30 min at 37°C or cross-linked to DNA by UV light at 254 nm for 10 min on ice, and samples were digested by trypsin. Resulting fragments were enriched by TiO 2 and analyzed by mass spectrometry (‘Materials and methods’ section). Identified amino acid positions cross-linked to DNA are denoted by a black cross. Positively charged residues and phosphorylation sites mutated in this study are noted in pink and orange, respectively, beneath the primary structure. ( B ) SDS-PAGE gel of purified human MORC2 protein (3 μg) stained with Coomassie blue stain. Contaminant band marked with an asterisk . ( C ) MORC2 DNA binding to DNA sequences of different AT/GC content as assessed by fluorescence anisotropy. Dephosphorylated MORC2 was titrated and incubated with 1 nM 5′-FAM-labeled 35 base pair duplex DNA with a high GC (71% GC content), high AT (31% GC content) or a random sequence with 49% GC content (‘Materials and methods’ section). Binding curves were fit with a single site quadratic binding equation. Error bars correspond to the standard deviation between three replicate experiments. ( D ) MORC2 binding to a 500 bp DNA substrate assessed by gel shift. Increasing concentrations of dephosphorylated MORC2 was incubated with 20 nM of duplex DNA and resolved on a 3–12% gradient Native PAGE gel (‘Materials and methods’ section). ( E ) MORC2 binding to a 1000 bp DNA substrate assessed by gel shift. Increasing concentrations of dephosphorylated MORC2 was incubated with 20 nM of duplex DNA and resolved on a 3–12% gradient Native PAGE gel (‘Materials and methods’ section). ( F ) MORC2 nucleic acid binding as measured by fluorescence anisotropy. Dephosphorylated MORC2 was titrated and incubated with 1 nM 5′-FAM-labeled 149 bp Widom 601 dsDNA, nucleosome, cruciform DNA, 35 nucleotide ssRNA and 35 base pair ssDNA (‘Materials and methods’ section). Binding curves were fit as in 1C except for the cruciform DNA which was fit with a Hill equation. Error bars correspond to the standard deviation between three replicate experiments. ( G ) MORC2 association with DNAs of varying topologies. Dephosphorylated MORC2 (150 nM) was incubated with 5′-FAM-labeled 35 base pair duplex DNA (1 nM), and positively supercoiled, negatively supercoiled, or relaxed plasmid DNA was titrated into the reactions (‘Materials and methods’ section). Binding curves were fit using an inhibition curve with a variable response. Error bars correspond to the standard deviation between three replicate experiments. ( H ) Assessment of DNA binding by dephosphorylated wild-type, aspartate mutant and 1–603 MORC2. MORC2 constructs were titrated and incubated with a FAM-labeled 35 base pair duplex DNA (1 nM) (‘Materials and methods’ section). Binding curves were fit as in 1C. Error bars correspond to the standard deviation between three replicate experiments.

Article Snippet: Primary antibodies used: β-ACT (Sigma-Aldrich #A5316, 1:10 000 dilution in 5% BSA in TBST), MORC2 (Bethyl Laboratories #A300-149A, 1:1000 dilution in 5% BSA in TBST), α-tubulin (Invitrogen #32–2500, 1:5000 dilution in 5% BSA in TBST), Lamin A/C (CST #2032, 1:1000 dilution in 5% BSA in TBST) and Histone H3 (EpiCypher #13–0001, 1:2500 dilution in 5% BSA in TBST).

Techniques: Purification, Mass Spectrometry, Phospho-proteomics, SDS Page, Staining, Binding Assay, Fluorescence, Incubation, Labeling, Sequencing, Standard Deviation, Gel Shift, Clear Native PAGE, Plasmid Preparation, Inhibition, Mutagenesis, Construct

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: Guide RNA sequences used to generate MORC2 knockout cells

Article Snippet: Primary antibodies used: β-ACT (Sigma-Aldrich #A5316, 1:10 000 dilution in 5% BSA in TBST), MORC2 (Bethyl Laboratories #A300-149A, 1:1000 dilution in 5% BSA in TBST), α-tubulin (Invitrogen #32–2500, 1:5000 dilution in 5% BSA in TBST), Lamin A/C (CST #2032, 1:1000 dilution in 5% BSA in TBST) and Histone H3 (EpiCypher #13–0001, 1:2500 dilution in 5% BSA in TBST).

Techniques: Knock-Out

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: PCR primers used to verify MORC2 knockout cells

Article Snippet: Primary antibodies used: β-ACT (Sigma-Aldrich #A5316, 1:10 000 dilution in 5% BSA in TBST), MORC2 (Bethyl Laboratories #A300-149A, 1:1000 dilution in 5% BSA in TBST), α-tubulin (Invitrogen #32–2500, 1:5000 dilution in 5% BSA in TBST), Lamin A/C (CST #2032, 1:1000 dilution in 5% BSA in TBST) and Histone H3 (EpiCypher #13–0001, 1:2500 dilution in 5% BSA in TBST).

Techniques: Knock-Out

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: MORC2 phosphorylation influences DNA binding and nuclear localization. ( A ) Phosphorylation reduces MORC2 affinity for DNA. Dephosphorylated, phosphorylated, phosphodead and phosphomimetic MORC2 were assessed for DNA binding using fluorescence anisotropy. MORC2 was titrated with FAM-labeled 35 base pair duplex DNA (1 nM) (‘Materials and methods’ section). Binding curves were fit using a quadratic binding equation. Error bars correspond to the standard deviation between three replicate experiments. ( B ) Effect of phosphorylation mutations on MORC2 localization in interphase HeLa cells. Representative confocal microscopy images of interphase HeLa cells overexpressing EGFP-wild-type MORC2, EGFP-MORC2 1–603, EGFP- MORC2 734–771, EGFP- phosphodead MORC2 and EGFP- phosphomimetic MORC2.

Article Snippet: Primary antibodies used: β-ACT (Sigma-Aldrich #A5316, 1:10 000 dilution in 5% BSA in TBST), MORC2 (Bethyl Laboratories #A300-149A, 1:1000 dilution in 5% BSA in TBST), α-tubulin (Invitrogen #32–2500, 1:5000 dilution in 5% BSA in TBST), Lamin A/C (CST #2032, 1:1000 dilution in 5% BSA in TBST) and Histone H3 (EpiCypher #13–0001, 1:2500 dilution in 5% BSA in TBST).

Techniques: Phospho-proteomics, Binding Assay, Fluorescence, Labeling, Standard Deviation, Confocal Microscopy

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: DNA binding by MORC2 reduces ATPase activity. ( A ) Assessment of MORC2 ATPase activity. Dephosphorylated wild-type, 1–603, DNA binding deficient aspartate mutant, and ATP hydrolysis deficient mutant E35A, and ATP binding deficient mutant N39A MORC2 (1 μM) were incubated with 1 mM ATP for 45 min at 37°C either in the presence or absence of a 35 base pair DNA (2 μM). Inorganic phosphate released was quantified by malachite green (‘Materials and methods’ section). Error bars correspond to the standard deviation between three replicate experiments. ( B ) MORC2 ATPase activity is reduced in the presence of DNA. Dephosphorylated MORC2 (1 μM) was incubated with 1 mM ATP for 45 min at 37°C with a titration of a 35 base pair duplex DNA. Inorganic phosphate released was quantified by malachite green (‘Materials and methods’ section). Data were fit to an inhibition curve. Error bars correspond to the standard deviation between three replicate experiments. ( C ) Michaelis–Menten analysis of MORC2 ATPase activity in the presence of DNA. Dephosphorylated MORC2 (1 μM) was incubated with an ATP titration for 45 min at 37°C in the presence of various concentrations of 35mer DNA (0–1 μM). Inorganic phosphate released was quantified by malachite green (‘Materials and methods’ section). Data were fit to a Michaelis–Menten model of enzyme kinetics. Error bars correspond to the standard deviation between three replicate experiments.

Article Snippet: Primary antibodies used: β-ACT (Sigma-Aldrich #A5316, 1:10 000 dilution in 5% BSA in TBST), MORC2 (Bethyl Laboratories #A300-149A, 1:1000 dilution in 5% BSA in TBST), α-tubulin (Invitrogen #32–2500, 1:5000 dilution in 5% BSA in TBST), Lamin A/C (CST #2032, 1:1000 dilution in 5% BSA in TBST) and Histone H3 (EpiCypher #13–0001, 1:2500 dilution in 5% BSA in TBST).

Techniques: Binding Assay, Activity Assay, Mutagenesis, Incubation, Standard Deviation, Titration, Inhibition

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: MORC2 homodimerizes via two distinct interfaces. ( A ) Dephosphorylated full-length MORC2 dimerizes in the presence and absence of AMP-PNP. Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) experiments with wild-type (monomer MW = 117 kDa) and 1–603 (monomer MW = 70 kDa) MORC2 in the presence or absence of 1 mM AMP-PNP. 2 mg/ml of MORC2 was applied to a WC-030 column (Wyatt Technology), and elution was monitored by absorption at 280 nm (left y -axis). MALS analysis of molecular weight is shown on the right y -axis, and average molecular weight calculations are shown across the center of each peak. ( B ) AlphaFold Multimer model of coiled coil 3 domain dimer. Chain A is colored gray and chain B is colored by pLDDT score. ( C ) MORC2 coiled coil 3 dimerizes in solution. SEC-MALS experiment performed with 1.5 mg/ml of purified MORC2 coiled coil 3 (monomer MW = 16 kDa) on a WC-010 column (Wyatt Technology). Elution was monitored by absorption at 280 nm (left y -axis). MALS analysis of molecular weight is shown on the right y -axis, and the average molecular calculation is shown across the center of the peak.

Article Snippet: Primary antibodies used: β-ACT (Sigma-Aldrich #A5316, 1:10 000 dilution in 5% BSA in TBST), MORC2 (Bethyl Laboratories #A300-149A, 1:1000 dilution in 5% BSA in TBST), α-tubulin (Invitrogen #32–2500, 1:5000 dilution in 5% BSA in TBST), Lamin A/C (CST #2032, 1:1000 dilution in 5% BSA in TBST) and Histone H3 (EpiCypher #13–0001, 1:2500 dilution in 5% BSA in TBST).

Techniques: Size-exclusion Chromatography, Multi-Angle Light Scattering, Molecular Weight, Purification

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: MORC2 can capture circular DNA substrates. ( A ) Schematic of assay to assess MORC2 capture of linear or circular DNA substrates. ( B ) Dephosphorylated, N-terminally-MBP-tagged wild-type, aspartate mutant and E35A MORC2 (600 nM) were incubated with supercoiled or linear pUC19 (100 nM). Samples were then added to amylose resin and washed in either low salt (50 mM NaCl) or high salt (400 mM NaCl) containing buffers before eluting the samples from the beads with maltose. Eluted samples were treated with proteinase K. DNA was resolved on a 1% (w/v) TAE agarose gel. Quantification of the band intensity from input and retained DNA bands normalized to the background are shown below each gel. Error bars correspond to the standard deviation between three replicate experiments. ( C ) Schematic of assay to assess MORC2 association with two circular DNA substrates. ( D ) A biotin-tagged DNA was conjugated to streptavidin magnetic beads to create a pseudo circular substrate. Dephosphorylated MORC2 (600 nM) was incubated with 20 μl of the beads in the presence or absence of 1 mM AMP-PNP. Supercoiled pBlueScript plasmid DNA (200 nM) was added, and 1 mM AMP-PNP was added or omitted before washing the beads with either low salt (50 mM NaCl) or high salt (400 mM NaCl) containing buffer. Samples were resuspended in 1X CutSmart buffer (New England Biolabs). DNA was released from the beads by digestion with ScaI and SbfI at 37°C for 1 h before proteinase K treatment. DNA was resolved on a 1% (w/v) TAE agarose gel. The intensity of the first DNA and second DNA substrate bands were quantified, normalized to the background and are presented as a ratio of second DNA:first DNA band intensity. Error bars correspond to the standard deviation between three replicate experiments. ( E ) Schematic of assay to assess MORC2 association with three circular DNA substrates. A biotin-tagged DNA was conjugated to streptavidin magnetic beads to create a pseudo-circular substrate. Dephosphorylated MORC2 (600 nM) was incubated with 20 μl of the beads. Supercoiled pBlueScript plasmid DNA (100 nM) and pBlueScript-601 plasmid DNA (100 nM) were added and 1 mM AMP-PNP was added or omitted before washing the beads with either low salt (50 mM NaCl) or high salt (400 mM NaCl) containing buffer. Samples were resuspended in 1X CutSmart buffer (New England Biolabs). DNA was released from the beads by digestion with ScaI and SbfI at 37°C for 1 h before proteinase K treatment. DNA was resolved on a 1% (w/v) TAE agarose gel.

Article Snippet: Primary antibodies used: β-ACT (Sigma-Aldrich #A5316, 1:10 000 dilution in 5% BSA in TBST), MORC2 (Bethyl Laboratories #A300-149A, 1:1000 dilution in 5% BSA in TBST), α-tubulin (Invitrogen #32–2500, 1:5000 dilution in 5% BSA in TBST), Lamin A/C (CST #2032, 1:1000 dilution in 5% BSA in TBST) and Histone H3 (EpiCypher #13–0001, 1:2500 dilution in 5% BSA in TBST).

Techniques: Mutagenesis, Incubation, Agarose Gel Electrophoresis, Standard Deviation, Magnetic Beads, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

doi: 10.1093/nar/gkae1273

Figure Lengend Snippet: DNA binding regulates MORC2 gene silencing activity in cells. ( A ) Schematic of RNAseq experiment. MORC2 constructs were added to HeLa cells lacking MORC2. MORC2 expression was induced for 48 h with doxycycline after which RNAs were extracted and sequenced. Volcano plots of RNAseq reads for overexpression of wild-type MORC2 versus control, overexpression of aspartate mutant MORC2 versus control, and wild-type MORC2 versus aspartate mutant MORC2 with spike normalization (‘Materials and methods’ section). Significant upregulated genes are shown in green, and significant downregulated genes are shown in purple from three biological replicates. Significant genes are classified as those that meet the fold change > 1.5 and FDR > 0.05 cutoffs. ( B ) Fold repression (-log 2 fold change) analysis of intronless and long-exon containing genes exhibiting a significant degree of repression after overexpression of wild-type MORC2 ( n = 75) in comparison to their fold repression after overexpression of aspartate mutant MORC2. Error bars correspond to the minimum and maximum values and the central bar represents the median value. Dotted lines connect genes between the two conditions. ( C ) Average fold repression (-log 2 fold change) of retrotransposon subfamilies as measured by RNAseq in cells with overexpression of wild-type or aspartate mutant MORC2 versus control cells using the TEtranscripts tool. Error bars represent the standard error between the elements in each subfamily (L1Hs n = 1, L1PA n = 30, L1M n = 86 and LTR-retrotransposons n = 347). ( D ) Model of how MORC2 engages DNA to promote compaction. MORC2 contains a DNA binding region between the GHKL domain and coiled coil 3 domain dimerization interfaces. DNA binding inside the lumen of the dimer communicates to the ATPase domain, to favor an ATP-bound homodimer conformation that is incompatible with ATP hydrolysis. MORC2 association with multiple DNA segments may allow MORC2 to bridge distal regions of DNA to contribute to compaction.

Article Snippet: Primary antibodies used: β-ACT (Sigma-Aldrich #A5316, 1:10 000 dilution in 5% BSA in TBST), MORC2 (Bethyl Laboratories #A300-149A, 1:1000 dilution in 5% BSA in TBST), α-tubulin (Invitrogen #32–2500, 1:5000 dilution in 5% BSA in TBST), Lamin A/C (CST #2032, 1:1000 dilution in 5% BSA in TBST) and Histone H3 (EpiCypher #13–0001, 1:2500 dilution in 5% BSA in TBST).

Techniques: Binding Assay, Activity Assay, Construct, Expressing, Over Expression, Control, Mutagenesis, Comparison