KDM4A's stability is regulated by the ubiquitin-proteasome system. (A) Immunoprecipitation (IP) and immunoblotting (WB) of lysates of HEK293T transiently transfected with the indicated vectors and treated with or without 10 μM MG132 for 4 h demonstrated that ubiquitylation of MYC-KDM4A is elevated in the presence of MG132. (B) Endogenous KDM4A protein levels were stabilized in a cycloheximide chase experiment performed in HEK293T cells when treated with MG132 and compared to control cells treated with vehicle (DMSO). HEK293T cells were treated with either DMSO or 10 μM MG132 for 4 h before the addition of 100 μg/ml of cycloheximide. Cells were harvested at the indicated times post-cycloheximide treatment, and lysates were immunoblotted using the indicated antibodies. (C) The indicated ubiquitin replacement U2OS cell lines were treated with either PBS or 4 μg/ml of doxycycline for 3 to 5 h, and cell lysates were immunoblotted using the indicated antibodies. Treatment of cells with doxycycline resulted in increased levels of endogenous KDM4A in cells with induced knockdown of endogenous ubiquitin as well as in induced cells with endogenous ubiquitin replaced with ubiquitin K48R mutant compared to their respective uninduced cells.

KDM4A interacts with components of the SCF^FBXO22 complex. (A) Mass spectrometry analyses of FLAG-HA-KDM4A and FLAG-HA-FBXO22 complexes independently showed that KDM4A interacts with FBXO22, SKP1, and CUL1 in HEK293T cells. (B) Ectopically expressed FLAG-FBXO22 immunoprecipitated with endogenous CUL1 and SKP1, affirming the interaction of FBXO22 with components of the SCF complex in HEK293T cells. Extracts of HEK293T cells transfected with either empty FLAG vector or vector expressing FLAG-FBXO22 were immunoprecipitated with anti-FLAG antibody, and immune complexes were blotted (WB) with the indicated antibodies. (C) Coexpression of FLAG-FBXO22 and MYC-KDM4A followed by IP and immunoblotting (WB) with the indicated antibodies verified the interaction of these two proteins together with CUL1 in HEK293T cells. (D) Endogenous IP of KDM4A in MLN4924-treated HEK293T cells confirmed the interaction of KDM4A and FBXO22 in HEK293T cells. HEK293T cells were treated with 1 μM MLN4924 for 4 h, and cell lysates were immunoprecipitated with either anti-KDM4A antibody or rabbit IgG. Immune complexes were blotted (WB) with either anti-KDM4A or anti-FBXO22 antibodies. (E) Eight FLAG-tagged F-box proteins were transiently expressed in HEK293T cells, and cell lysates were immunoprecipitated with anti-HA antibody. Immune complexes were blotted with the indicated antibodies to examine the interaction of each F-box with endogenous KDM4A. (F) Based on cell fractionation and subsequent immunoblotting of the various fractions with the indicated antibodies, endogenous FBXO22 was found in both the nucleus and cytoplasm, and endogenous KDM4A was found predominantly in the nucleus of HeLa cells. (G) HeLa cells were subjected to either double thymidine block to obtain G₁/S cells or nocodazole treatment to obtain G₂/M cells. The cells were then harvested, and immunoprecipitation was performed using either anti-KDM4A antibody or rabbit IgG. Subsequent immunoblotting with the indicated antibodies showed that the interaction of endogenous FBXO22 and KDM4A is similar in both G₁/S and G₂/M phases. (H) HeLa cells were arrested either in G₁/S or G₂/M phase by double thymidine block or by double thymidine block followed by release into nocodazole for 10 h to harvest late G₂-to-G₂/M cells with intact nuclei. When we used cell fractionation and subsequent immunoblotting of the various fractions with the indicated antibodies, endogenous FBXO22 did not display a significant difference in cellular localization at G₁/S and G₂/M phases.

The interaction of FBXO22 and KDM4A is mediated by their FIST-C and Jmj domains, respectively. (A) Schematic diagram of the different FLAG-HA-tagged KDM4A deletion mutants. (B) Immunoprecipitation (IP) of endogenous FBXO22 with either full-length KDM4A or various KDM4A mutants demonstrated a requirement for the JmjN and JmjC domains for interaction. Cell lysates of HEK293T cells transiently expressing either full-length FLAG-HA-KDM4A or various FLAG-HA-KDM4A mutants were immunoprecipitated with anti-HA antibody, and immune complexes were blotted with anti-FBXO22 antibody. WCE, whole-cell extract. (C) Schematic representing the different FLAG-HA-tagged FBXO22 deletion mutants. (D) Immunoprecipitation of MYC-tagged KDM4A with either full-length FBXO22 or various FBXO22 mutants revealed a requirement for the FIST-C domain. Cell lysates of HEK293T cells transiently expressing either full-length FLAG-HA FBXO22 or various recombinant FLAG-HA FBXO22 mutants, together with MYC-KDM4A, were immunoprecipitated with anti-HA antibody, and immune complexes were blotted with anti-MYC antibody.

SCF^FBXO22 regulates KDM4A abundance and ubiquitylation in cells and promotes KDM4A ubiquitylation in vitro. (A) Steady-state levels of KDM4A are increased in HeLa cells treated with MLN4924 (cullin neddylation inhibitor) and bortezomib (proteasomal inhibitor). HeLa cells were treated with either 1 μM MLN4924 or 500 nM bortezomib for 4 h, and cell lysates were immunoblotted (WB) with the indicated antibodies. (B) Stability of KDM4A is increased in HEK293T cells treated with MLN4924 compared to control DMSO-treated cells, as demonstrated in a cycloheximide chase experiment. HEK293T cells were treated with either DMSO or 1 μM MLN4924 for 4 h before the addition of 100 μg/ml of cycloheximide. Cells were harvested at the indicated time points after cycloheximide treatment, and lysates were immunoblotted using the indicated antibodies. (C) Overexpression of CUL1^DN increases endogenous levels of KDM4A in HEK293T cells. HEK293T cells were transiently transfected with either empty FLAG vector or vector expressing FLAG-CUL1^DN for 48 h, and lysates were immunoblotted with the indicated antibodies. (D) Depletion of FBXO22 using 3 independent siRNAs increased KDM4A levels in both HEK293T and HeLa cells. Cell lysates of HEK293T and HeLa cells transfected with the indicated siRNAs for 72 h were immunoblotted with the indicated antibodies. (E) Depletion of FBXO22 led to stabilization of KDM4A in a cycloheximide chase experiment. HEK293T cells were transiently transfected with either control or FBXO22 siRNAs for 60 h before the addition of 100 μg/ml cycloheximide. Cells were harvested at the indicated time points post-cycloheximide treatment, and lysates were immunoblotted using the indicated antibodies. (Right panel) Graphical representation of data obtained from three independent experiments, using three different FBXO22 siRNAs, as quantified with ImageJ software (NIH). (F) Ectopically expressed MYC-FBXO22 led to a dose-dependent decrease in steady-state KDM4A levels, which could be rescued by the addition of MG132. HEK293T cells were transiently transfected with the indicated amounts of vector expressing MYC-FBXO22, and lysates were immunoblotted with the indicated antibodies to determine levels of endogenous KDM4A. MG132 at 10 μM was added to the last sample, as indicated, for 4 h. (Lower panel) Graphical representation of data obtained from two independent experiments as quantified by using ImageJ software. (G) Expression of FBXO22 but not the F-box deletion mutant led to a significant increase in the polyubiquitylation of MYC-KDM4A. Vectors expressing the indicated proteins were transiently transfected into HEK293T cells. At 48 h after transfection, cells were treated with 10 μM MG132 for 4 h, and cell lysates were immunoprecipitated with anti-HA antibody. Immune complexes were blotted with the indicated antibodies. (H) Ubiquitylation of KDM4A by HEK293T-purified SCF^FBXO22 in vitro. (Lower panel) Graphical representation of the average of the relative levels of ubiquitylation of MYC-KDM4A in each sample for two independent in vitro ubiquitylation experiments as quantified with ImageJ software. Error bars represent the standard deviations from averages.

Depletion of FBXO22 leads to global changes in KDM4A-targeted histone methylation marks. (A and B) Depletion of FBXO22 with three independent siRNAs led to global decreases in KDM4A-targeted histone H3K9 and H3K36 trimethylation marks and a corresponding increase in global H3K9 and H3K36 monomethylation. HEK293T cells were transfected with the indicated siRNAs for 72 h and harvested. Half the cells for each siRNA transfection were lysed with regular lysis buffer and used to probe for FBXO22 and KDM4A levels, while the remainder was acid extracted to enrich for histones and immunoblotted for the various methylation marks using the indicated antibodies. Quantification of Western blotting (WB) images was performed using ImageJ software (NIH) and is presented in graphical form in panel B. The data in panel B represent averages of two independent experiments, and error bars represent the standard deviations from these averages. (C) Overexpression of KDM4A induces global depletion of H3K9 and H3K36 trimethylation marks. HEK293T cells were transfected with the indicated amounts of FLAG-HA-KDM4A for 48 h and harvested. Cells were processed, and lysates were used for immunoblotting as described for panel A.

Depletion or overexpression of FBXO22 affects mRNA levels and histone methylation levels on a KDM4A target gene, ASCL2. (A) Position of the JAR in the ASCL2 promoter relative to the transcriptional start site. (B) ChIP-qPCR showed that depletion of FBXO22 in HEK293T cells using two independent siRNAs led to depletion of (i) histone H3K9me3 and (ii) H3K36me3, but not (iii) H3K4me3 levels, at the KDM4A target region on ASCL2 but not at a nontargeted region on chromosome 4 pericentric heterochromatin. FBXO22 at 30 nM and control siRNAs were transfected into HEK293T cells and harvested 72 h posttransfection. A small aliquot of cells was used to verify the knockdown efficiency by immunoblotting (>90% knockdown of FBXO22 protein). After sonication and lysate preclearing, the lysate was used for detection of H3, H3K4me3, H3K9me3, H3K36me3, and Rb IgG ChIP. Typically a >20-fold enrichment from Rb IgG ChIP (data not shown) was observed at all regions sampled. Data represent the averages of three independent experiments, and error bars represent the standard deviations from these averages. (C) Depletion of KDM4A using 3 independent shRNAs that produce >95% knockdown of KDM4A protein levels results in ∼6-fold derepression of ASCL2 transcription levels as measured by qPCR. HE293T cells were infected with lentivirus containing the various shRNAs and selected under 1 μg/ml puromycin for >72 h. Protein lysates were used for immunoblotting, and TRIzol extracts were used for qPCR. Error bars for qPCR results represent the standard deviations from the means of two independent experimens. (D) Ectopic expression of KDM4A, but not its catalytic mutant (H188A), leads to repression of ASCL2 transcription in a dose-dependent manner. Two micrograms of FLAG-HA empty vector (Ctl), 0.5 μg or 2 μg of FLAG-HA-KDM4A, or FLAG-HA-KDM4AH188A was transfected into subconfluent HEK293T cells. Cells were harvested 48 h posttransfection and processed as described for panel C. Error bars represent the standard deviations from the means (n = 2). (E) Ectopic expression of MYC-FBXO22 resulted in a dose-dependent activation of ASCL2 transcription levels. Two micrograms of MYC empty vector or 0.5 μg, 1 μg, or 2 μg MYC-FBXO22 was transfected into HEK293T cells and processed as described for panel C. Error bars represent the standard deviations from the means (n = 2). (F) Depletion of FBXO22 by RNAi resulted in repression of ASCL2 transcription levels. HEK293T cells were transfected with 30 nM siRNA, harvested 72 h posttransfection, and processed as described for panel C. Error bars represent the standard deviations from the means (n = 2).

Proposed model for how posttranslational turnover of KDM4A by the SCF^FBXO22-proteasome pathway leads to histone methylation changes and the target gene transcriptional effects. KDM4A is thought to be recruited to specific gene loci at least partly by its tandem Tudor domains, which bind H3K4me3 marks on chromatin. The catalytic JmjC domain is then able to mediate demethylation of H3K9me3/2 as well as H3K36me3/2 marks, although it is not clear if this can happen on the same histone or nucleosome. Upon an appropriate signal for degradation, KDM4A may be delocalized from its target sites and then be targeted by the SCF^FBXO22 ubiquitin ligase complex, as illustrated by arrow A. This interaction is mediated by the JmjN/JmjC domain of KDM4A and the FIST-C domain on FBXO22. Polyubiquitylated KDM4A is then targeted for degradation by the proteasome. In the absence of a degradation signal or when components of the SCF^FBXO22 complex are experimentally depleted (arrow B), KDM4A can accumulate to levels sufficient to demethylate its target marks. In the case of FBXO22 RNAi, where there is an overaccumulation of KDM4A, indiscriminate demethylation of its target marks may ensue over many gene loci, leading to global loss of H3K9me3 and H3K36me3 marks while leaving H3K9me1 and H3K36me1 marks in its wake. In the case of ASCL2, loss of H3K9me3 and H3K36me3 at the promoter region leads to transcriptional repression.