Journal: Scientific Reports

Article Title: Histone demethylase KDM4D cooperates with NFIB and MLL1 complex to regulate adipogenic differentiation of C3H10T1/2 mesenchymal stem cells

doi: 10.1038/s41598-020-60049-8

Figure Lengend Snippet: KDM4D is required for the adipogenic differentiation of C3H10T1/2 mesenchymal stem cells. ( a–c ). Knockdown of KDM4D by the shRNA inhibits the adipogenesis of C3H10T1/2 cells. C3H10T1/2 cells and two independently established KDM4D-knockdowned cells (shKDM4D-1 and shKDM4D-2) were grown to confluence and adipogenic differentiation was induced by MDI hormonal treatment. ( a ) Schematic diagram depicting the sites of Kdm4d mRNA targeted by shRNA (upper). Sh1 targets 3′-UTR (shKDM4D-1) and Sh2 targets the coding region of Kdm4d mRNA (shKDM4D-2). Oil Red O staining at day eight after the induction of differentiation. The insets present plate images after ORO staining (lower). ( b ) Immunoblot analysis of KDM4D and key adipogenic markers before and after induction of differentiation. The total extracts were prepared from the cells at indicated time points (day 0; before MDI treatment) and subjected to immunoblot analysis to detect KDM4D, PPARγ, C/EBPα, and aP2. Actin was used as a loading control. ( c ) RT-qPCR analysis of adipogenic gene expression before and after the induction of differentiation. The total RNAs were isolated from control, shKDM4D-1, and shKDM4D-2 cells at the indicated time points, and the relative mRNA levels of Cebpb , Pparg , Cebpa , and aP2 were measured by RT-qPCR. The mRNA levels were first normalized to the mRNA level of GAPDH and the data are presented as the ratio of the mRNA level at each time point (after induction) to the mRNA level at day 0 (before induction). Quantitative PCR (qPCR) data are representative of at least three independent experiments and are presented as mean ± SD. * p < 0.05; ** p < 0.01. (d) Rescue of differentiation by exogenous FLAG-KDM4D. The control cells and shKDM4D-1 cells, in which shRNA targets 3′ UTR of Kdm4d , were infected with a retrovirus expressing empty vector or FLAG-KDM4D. (Left) Oil Red O staining at day eight after the induction of differentiation. (Right) Immunoblot analysis of KDM4D (with α-FLAG and α-KDM4D), PPARγ, and aP2 before (day 0) and after (day 8) induction of differentiation. Actin was used as a loading control. (e) Overexpression of KDM4D had no significant effect on the adipogenic differentiation of C3H10T1/2 MSCs. The cells were infected with the retrovirus expressing empty vector or FLAG-KDM4D, followed by the induction of adipogenic differentiation. (Left) Oil Red O staining at day eight after the induction of differentiation. (Right) The total extracts were prepared from the cells at the indicated time points and subjected to immunoblot analysis of KDM4D, PPARγ, and aP2. Actin was used as a loading control. The images of immunoblot analysis (b,d,e) were cropped from different gels due to similar molecular weight but all panels in each figure are from the same experiment. Unprocessed images are provided in the supplementary information.

Article Snippet: The full-length cDNAs used in this study were either amplified directly from mouse cDNA ( Kdm4d ) or purchased from Origene ( Nfib : MR206682, Rockville, MD, USA), Addgene ( Kdm4d : #61553, Cebpa : #66978, Cebpb : #66979, Wdr5 : #15552, Watertown, MA, USA), and Korea Human Gene Bank ( Kdm4a : mMU005272, Kdm4c : mMU003529, Pparg : mMU001014, Daejeon, Korea).

Techniques: Knockdown, shRNA, Staining, Western Blot, Control, Quantitative RT-PCR, Gene Expression, Isolation, Real-time Polymerase Chain Reaction, Infection, Expressing, Plasmid Preparation, Over Expression, Molecular Weight

Journal: Scientific Reports

Article Title: Histone demethylase KDM4D cooperates with NFIB and MLL1 complex to regulate adipogenic differentiation of C3H10T1/2 mesenchymal stem cells

doi: 10.1038/s41598-020-60049-8

Figure Lengend Snippet: Exogenous expression of PPARγ or C/EBPα rescues adipogenic differentiation in KDM4D-depleted cells. The control and shKDM4D cells were infected with retrovirus expressing empty vector or FLAG-tagged C/EBPβ ( a ), PPARγ ( b ), or C/EBPα ( c ), followed by the induction of differentiation for eight days. (a) Exogenous expression of FLAG-C/EBPβ does not rescue adipogenic differentiation in shKDM4D cells. Oil Red O staining at day eight after the induction of differentiation (upper) and immunoblot analysis of FLAG-C/EBPβ and aP2 (lower). (b,c) Exogenous expression of FLAG-PPARγ ( b ) or FLAG-C/EBPα ( c ) rescues adipogenic differentiation in shKDM4D cells. Oil Red O staining at day 8 after induction of differentiation (upper) and immunoblot analysis of FLAG-PPARγ ( b ), FLAG-C/EBPα ( c ), and aP2 (lower). (d) RT-qPCR analysis of aP2 in the control and shKDM4D cells infected with the retrovirus expressing empty vector or FLAG-C/EBPβ (left), FLAG-PPARγ (middle) or FLAG-C/EBPα (right). The mRNA levels of aP2 were first normalized to the mRNA level of GAPDH , and the data are presented as the ratio of mRNA level at each time point to the mRNA level in the control cells at day 0 (before induction). The quantitative PCR data are representative of at least three independent experiments and presented as mean ± SD. ** p < 0.01. The images of immunoblot analysis ( a–c ) were cropped from different gels because of similar molecular weight. All panels in each figure are from the same experiment. Unprocessed images are provided in the supplementary information.

Article Snippet: The full-length cDNAs used in this study were either amplified directly from mouse cDNA ( Kdm4d ) or purchased from Origene ( Nfib : MR206682, Rockville, MD, USA), Addgene ( Kdm4d : #61553, Cebpa : #66978, Cebpb : #66979, Wdr5 : #15552, Watertown, MA, USA), and Korea Human Gene Bank ( Kdm4a : mMU005272, Kdm4c : mMU003529, Pparg : mMU001014, Daejeon, Korea).

Techniques: Expressing, Control, Infection, Plasmid Preparation, Staining, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Molecular Weight

Journal: Scientific Reports

Article Title: Histone demethylase KDM4D cooperates with NFIB and MLL1 complex to regulate adipogenic differentiation of C3H10T1/2 mesenchymal stem cells

doi: 10.1038/s41598-020-60049-8

Figure Lengend Snippet: KDM4D interacts with NFIB transcription factor and MLL1 H3K4 methyltransferase complex. ( a ) KDM4D was co-purified with NFIB, components of MLL complex, and components of NuRD remodeling complex. C3H10T1/2 cells were infected with the retrovirus expressing empty vector or FLAG-KDM4D and nuclear extracts were prepared as described in methods. KDM4D interacting proteins were purified with FLAG-M2 agarose and subjected to mass spectrometric analysis. Shown is a silver staining image of FLAG-KDM4D and co-purified proteins. ( b,c ) KDM4D interacts with NFIB in C3H10T1/2 cells. The cells were infected with the retrovirus expressing empty vector, FLAG-KDM4D ( b ) or FLAG-NFIB ( c ). The protein extracts were immunoprecipitated with FLAG-M2 agarose and the interactions were confirmed by immunoblot analysis using α-NFIB in ( b ) and α-KDM4D in ( c ). (d,e) KDM4D and NFIB interact with MLL1 histone methyltransferase complex in C3H10T1/2 cells. The cells were infected with the retrovirus expressing empty vector, FLAG-KDM4D ( d ), or FLAG-NFIB ( e ). The protein extracts were immunoprecipitated with FLAG-M2 agarose, and the interactions were examined by immunoblot analysis using the indicated antibodies. Actin was used as a loading control. (f) KDM4D, but not other members of the KDM4 family of proteins, interacts with NFIB. C3H10T1/2 cells were infected with the retrovirus expressing empty vector or indicated FLAG-KDM4 proteins (KDM4A - KDM4D). The protein extracts were immunoprecipitated with FLAG-M2 agarose, followed by immunoblot analysis to determine the interaction with endogenous NFIB. The asterisk indicates non-specific band. ( g ) Schematic diagrams of KDM4D (upper), NFIB (lower), and their deletion mutants. JmjN and JmjC domains of KDM4D (upper) and N-terminal DNA binding domain and transcription modulation domain of NFIB (lower) are shown as gray boxes. ( h ) JmjC domain of KDM4D mediates the interaction with NFIB. Human 293 T cells were co-transfected with plasmids containing full-length FLAG-NFIB and indicated HA-KDM4D (FL; full-length, numbered; deletion mutants). Half of the protein extracts were immunoprecipitated with FLAG-M2 agarose and the other half were immunoprecipitated with α-HA antibody. The interactions between full-length NFIB and KDM4D (and its deletion mutants) were determined by immunoblot analysis using α-HA (for FLAG IP) and α-FLAG (for HA IP). The asterisk indicates the immunoglobulin light chain band. (i) N-terminal domain of NFIB is required for the interaction with KDM4D. Human 293 T cells were co-transfected with plasmids containing full-length FLAG-KDM4D and indicated HA-NFIB constructs (FL; full-length, numbered; deletion mutants). The protein extracts were immunoprecipitated with α-HA antibody. Interactions between the full-length KDM4D and NFIB (and its deletion mutants) were determined by immunoblot analysis using α-FLAG and α-KDM4D. The asterisk indicates the immunoglobulin heavy chain band. The images of immunoblot analysis shown were cropped from different gels to optimize separation (MLL proteins) and distinguish the proteins with similar molecular weight. Unprocessed images are provided in the supplementary information.

Article Snippet: The full-length cDNAs used in this study were either amplified directly from mouse cDNA ( Kdm4d ) or purchased from Origene ( Nfib : MR206682, Rockville, MD, USA), Addgene ( Kdm4d : #61553, Cebpa : #66978, Cebpb : #66979, Wdr5 : #15552, Watertown, MA, USA), and Korea Human Gene Bank ( Kdm4a : mMU005272, Kdm4c : mMU003529, Pparg : mMU001014, Daejeon, Korea).

Techniques: Purification, Infection, Expressing, Plasmid Preparation, Silver Staining, Immunoprecipitation, Western Blot, Control, Binding Assay, Transfection, Construct, Molecular Weight

Journal: Scientific Reports

Article Title: Histone demethylase KDM4D cooperates with NFIB and MLL1 complex to regulate adipogenic differentiation of C3H10T1/2 mesenchymal stem cells

doi: 10.1038/s41598-020-60049-8

Figure Lengend Snippet: KDM4D, NFIB, and MLL1 complex work together in regulating adipogenic differentiation. ( a,b ) NFIB and MLL1 are required for the adipogenic differentiation of C3H10T1/2 cells. The control and three different KD cells (shKDM4D, shNFIB and shMLL1) were grown to confluence, and adipogenic differentiation was induced by MDI hormonal treatment. ( a ) Oil Red O staining at day eight after the induction of differentiation. ( b ) RT-qPCR analysis of adipogenic gene expression before and after the induction of differentiation. The total RNAs were isolated from the control, shKDM4D, shNFIB, and shMLL1 cells before (day 0) and after (day 8) the induction of differentiation. The relative mRNA levels of Pparg , Cebpa , and aP2 were measured by RT-qPCR. The mRNA levels were normalized to the mRNA level of GAPDH and the data are presented as the ratio of the mRNA level at each time point to the mRNA level in the control cells at day 0. Quantitative PCR data are representative of at least three independent experiments and presented as mean ± SD. ** p < 0.01. ( c–f ) KDM4D, NFIB, and MLL1 are required for adipogenic gene expression. The cells were collected before (day 0) and after (day 5) the induction of differentiation for RNA-seq analysis. ( c ) RNA-seq heatmap depicting the changes in expression of the genes (upregulated during adipogenic differentiation of C3H10T1/2 cells) in the control, shKDM4D, shNFIB, and shMLL1 cells. The color intensity scale was included at the bottom of the heatmap. The threshold for up-regulation is 2.0 fold. ( d ) Schematic diagram of the identification of KDM4D-, NFIB-, and MLL1-dependent genes (left) and Venn diagram of the genes affected by KDM4D, NFIB, and MLL1 depletion (lower than the control cells at day 5). ( e ). Gene ontology (GO) analysis (upper) and KEGG pathway analysis (lower) of the genes defined as commonly affected genes in ( d ). ( f ) Venn diagram of the genes identified as KDM4D/NFIB/MLL1-dependent and direct targets of PPARγ and C/EBPα. ( g,h ) KDM4D and NFIB bind to the promoter of Cebpa , and Pparg genes. C3H10T1/2 cells were infected with a retrovirus expressing empty vector or FLAG-KDM4D ( g ) and FLAG-NFIB ( h ). Chromatins prepared from the cells were precipitated with FLAG-M2 agarose, and quantitative PCR (qPCR) analysis was performed to assess the binding to the promoter and distal region of the indicated genes. For the relative ChIP signal, the % input was calculated for each sample, and data are presented as the ratio of the % input in FLAG-KDM4D-infected cells to the % input in the control cells. ( i ) MLL1 binds to the promoter of Cebpa , and Pparg genes. Chromatins prepared from the cells before (day 0) and after (day 5) the induction of differentiation were precipitated with IgG or α-MLL1 antibody. qPCR analysis was performed as in ( g,h ). For the relative ChIP signal, the % input was calculated for each sample and data are presented as the ratio of the % input (α-MLL1) to the % input (IgG). Quantitative PCR data shown in ( g – i ) are representative of at least three independent experiments and are presented as mean ± SD. * p < 0.05; ** p < 0.01.

Article Snippet: The full-length cDNAs used in this study were either amplified directly from mouse cDNA ( Kdm4d ) or purchased from Origene ( Nfib : MR206682, Rockville, MD, USA), Addgene ( Kdm4d : #61553, Cebpa : #66978, Cebpb : #66979, Wdr5 : #15552, Watertown, MA, USA), and Korea Human Gene Bank ( Kdm4a : mMU005272, Kdm4c : mMU003529, Pparg : mMU001014, Daejeon, Korea).

Techniques: Control, Staining, Quantitative RT-PCR, Gene Expression, Isolation, Real-time Polymerase Chain Reaction, RNA Sequencing, Expressing, Infection, Plasmid Preparation, Binding Assay

Journal: Scientific Reports

Article Title: Histone demethylase KDM4D cooperates with NFIB and MLL1 complex to regulate adipogenic differentiation of C3H10T1/2 mesenchymal stem cells

doi: 10.1038/s41598-020-60049-8

Figure Lengend Snippet: KDM4D requires MLL1 to interact with NFIB but is dispensable for the interaction between NFIB and MLL1 complex as well as their binding to the Pparg and Cebpa promoters. Control and KD cells (shNFIB, shMLL1, and shKDM4D, respectively) were infected with the retrovirus expressing empty vector or indicated FLAG-tagged proteins. The total protein extracts and chromatins were prepared after the induction of differentiation (day 5), followed by the immunoprecipitation assay and ChIP-qPCR analysis. ( a–c ) NFIB is required for the interaction between KDM4D and MLL1 complex and their binding to the Pparg and Cebpa promoters. ( a ) Immunoblot analysis of the interaction between FLAG-KDM4D and MLL1 complex in the control and shNFIB cells. ( b,c ) ChIP-qPCR analysis of FLAG-KDM4D ( b ) and MLL1 ( c ) bindings to the Cebpb , Cebpa , and Pparg promoters in the control and shNFIB cells. ( d–f ) MLL1 is necessary for the interaction between KDM4D and NFIB and their binding to the target promoters. ( d ) Immunoblot analysis of the interaction between FLAG-KDM4D and NFIB in the control and shMLL1 cells. ( e,f ) ChIP-qPCR analysis of FLAG-NFIB ( e ) and FLAG-KDM4D ( f ) bindings to the Cebpb , Cebpa , and Pparg promoters in the control and shMLL1 cells. ( g–i ) KDM4D is dispensable for the interaction between NFIB and MLL1 complex and their bindings to the target promoters. ( g ) Immunoblot analysis of the interaction between FLAG-NFIB and MLL1 complex in the control and shMLL1 cells. ( h,i ) ChIP-qPCR analysis of FLAG-NFIB ( h ) and MLL1 ( i ) bindings to the Cebpb , Cebpa , and Pparg promoters in the control and shKDM4D cells. For the relative ChIP signal, the % input was calculated for each sample. Data shown in figures ( b , e , f , h ) were presented as the ratio of the % input in the cells expressing FLAG-KDM4D ( b,f ) or FLAG-NFIB (e and h) to the % input in the control cells infected with retrovirus expressing empty vector. Data shown in figures ( c,i ) were presented as the ratio of the % input (α-MLL1) to % input (IgG) in the control cells. Quantitative PCR data in all figures are representative of three independent experiments and are presented as mean ± SD. * p < 0.05; ** p < 0.01. The images of immunoblot analysis shown ( a,d,g ) were cropped from different gels to optimize separation (MLL proteins) and to distinguish proteins with a similar molecular weight. All panels in each figure are from the same experiment. Unprocessed images are provided in the supplementary information.

Article Snippet: The full-length cDNAs used in this study were either amplified directly from mouse cDNA ( Kdm4d ) or purchased from Origene ( Nfib : MR206682, Rockville, MD, USA), Addgene ( Kdm4d : #61553, Cebpa : #66978, Cebpb : #66979, Wdr5 : #15552, Watertown, MA, USA), and Korea Human Gene Bank ( Kdm4a : mMU005272, Kdm4c : mMU003529, Pparg : mMU001014, Daejeon, Korea).

Techniques: Binding Assay, Control, Infection, Expressing, Plasmid Preparation, Immunoprecipitation, ChIP-qPCR, Western Blot, Real-time Polymerase Chain Reaction, Molecular Weight

Journal: Scientific Reports

Article Title: Histone demethylase KDM4D cooperates with NFIB and MLL1 complex to regulate adipogenic differentiation of C3H10T1/2 mesenchymal stem cells

doi: 10.1038/s41598-020-60049-8

Figure Lengend Snippet: KDM4D coordinates H3K4me3/H3K9me3 to regulate the adipogenic differentiation of C3H10T1/2 mesenchymal stem cells. ( a,b ) KDM4D is required for the tri-methylation of H3K4 as well as the demethylation of tri-methylated H3K9 at the Cebpa and Pparg promoters in adipogenic differentiation. Chromatins prepared from control cells and three KD cells (shKDM4D, shNFIB, and shMLL1) before (day 0) and after (day 5) the induction of differentiation were precipitated with IgG ( a,b ), α-H3K4me3 ( a ), and α-H3K9me3 ( b ) antibodies. The data are presented as the % input of indicated antibodies and the dotted lines indicate the % input of IgG. Quantitative PCR data in all figures are representative of three independent experiments and are presented as mean ± SD. * p < 0.05; ** p < 0.01. ( c ) A model for the roles of KDM4D, NFIB, and MLL1 complex in the transcription activation of Cebpa and Pparg during adipogenic differentiation.

Article Snippet: The full-length cDNAs used in this study were either amplified directly from mouse cDNA ( Kdm4d ) or purchased from Origene ( Nfib : MR206682, Rockville, MD, USA), Addgene ( Kdm4d : #61553, Cebpa : #66978, Cebpb : #66979, Wdr5 : #15552, Watertown, MA, USA), and Korea Human Gene Bank ( Kdm4a : mMU005272, Kdm4c : mMU003529, Pparg : mMU001014, Daejeon, Korea).

Techniques: Methylation, Control, Real-time Polymerase Chain Reaction, Activation Assay