Journal: bioRxiv

Article Title: KDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity

doi: 10.1101/561571

Figure Lengend Snippet: (A) A schematic illustrating protein domain architecture for KDM2A/B long (LF) and short isoforms (SF). (B) A schematic of the Kdm2a/b-JmjC fl/fl system in which addition of tamoxifen (OHT) leads to removal of KDM2 long isoforms. (C) Western blot analysis for KDM2A and KDM2B in K dm2a/b-JmjC fl/fl mESCs before (UNT) and after 96 hours of tamoxifen (OHT) treatment. BRG1 is shown as a loading control for both blots. Asterisks indicate non-specific bands. (D) Heatmaps of H3K36me2 enrichment (ChIP-seq) in K dm2a/b-JmjC fl/fl mESCs before (UNT) and after addition of tamoxifen (OHT), for CGI-associated (n=14106) and non-CGI-associated (n=6527) gene promoters. H3K36me2 signal was normalised to H3 ChIP-seq to control for any alterations in nucleosome density. (E) A metaplot of normalised H3K36me2 ChIP-seq signal at CGI-associated or non-CGI-associated gene promoters in K dm2a/b-JmjC fl/fl mESCs, before (UNT) and after tamoxifen treatment (OHT). (F) A metaplot of normalised H3K36me2 ChIP-seq signal at intragenic CGIs in K dm2a/b-JmjC fl/fl mESCs, before (UNT) and after tamoxifen treatment (OHT). (G) Metaplots showing normalised H3K36me2 ChIP-seq signal throughout the gene body before (UNT) and after tamoxifen treatment (OHT), for CGI-associated genes separated into quartiles according to their expression level in K dm2a/b-JmjC fl/fl mESCs (Q1 < Q2 < Q3 < Q4). Genes were scaled to the same length and aligned at their TSS and TES. (H) A boxplot showing fold change in normalised H3K36me2 ChIP-seq signal following tamoxifen treatment, for the CGI-associated gene quartiles shown in (G) and for non-CGI-associated genes. (I) A metaplot showing H3K36me3 enrichment throughout the gene body for the gene sets shown in (G) .

Article Snippet: Chromatin was pre-cleared for 1 hour with either protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) or protein A agarose beads (Repligen, for histone or RNAPII ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA.

Techniques: Western Blot, ChIP-sequencing, Expressing

Journal: bioRxiv

Article Title: KDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity

doi: 10.1101/561571

Figure Lengend Snippet: (A) A schematic representation of the Kdm2a/b-JmjC fl/fl mESC line, in which loxP sites were inserted into the Kdm2a and Kdm2b genes flanking exons that encode the JmjC domain. (B) ChIP-qPCR analysis showing KDM2A (upper panel) and KDM2B (lower panel) enrichment relative to input in Kdm2a/b-JmjC fl/fl mESCs before (UNT) and after tamoxifen treatment (OHT). Error bars show standard error of the mean of three biological replicates. (C) Quantitation of western blots of histone extract from Kdm2a/b-JmjCfl/fl mESCs, before (UNT) and after 96 hours of tamoxifen treatment (OHT). Signal is normalised to histone H4 and is represented relative to average UNT signal. Error bars show standard deviation of three biological replicates. Significance was tested using a Student’s T-test (non-significant (ns) if p > 0.05). (D) Representative western blots. Histone H4 is shown as a loading control. (E) Boxplots showing CpG density (left) and size (right) of intragenic and promoter-associated CGIs. (F) A metaplot of cnRNA-seq signal in K dm2a/b-JmjC fl/fl mESCs, for CGI-associated genes separated into quartiles according to their expression level and for non-CGI-associated genes. Genes were scaled to the same length and aligned at their TSS and TES.

Article Snippet: Chromatin was pre-cleared for 1 hour with either protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) or protein A agarose beads (Repligen, for histone or RNAPII ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA.

Techniques: Quantitation Assay, Western Blot, Standard Deviation, IF-P, Expressing

Journal: bioRxiv

Article Title: KDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity

doi: 10.1101/561571

Figure Lengend Snippet: (A) An MA-plot showing log2 fold change in the accessibility (cATAC-seq) of CGI-associated gene promoters in K dm2a/b-JmjC fl/fl mESCs following tamoxifen treatment. No promoters significantly changed in accessibility (p-adj < 0.05 and > 1.4-fold). (B) An MA-plot showing log2 fold change in gene expression (cnRNA-seq) in K dm2a/b-JmjC fl/fl mESCs following tamoxifen treatment. The number of genes with significantly increased or decreased expression (p-adj < 0.05 and > 1.4-fold) is shown in red and density of gene expression changes is shown on the right. (C) A scatter plot comparing the log2 fold change in gene expression (cnRNA-seq) with the log2 fold change in normalised H3K36me2 ChIP-seq signal for CGI-associated genes following tamoxifen treatment of Kdm2a/b-JmjC fl/fl mESCs. The solid line shows the linear regression, and the coefficient of determination (R 2 ) and Spearman correlation coefficient (Cor) are annotated. (D) Boxplots showing the log2 fold change in cnRNA-seq signal (left) and normalised H3K36me2 ChIP-seq signal (right) for CGI-associated genes grouped into quintiles based on their change in expression following tamoxifen treatment of Kdm2a/b-JmjC fl/fl mESCs.

Article Snippet: Chromatin was pre-cleared for 1 hour with either protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) or protein A agarose beads (Repligen, for histone or RNAPII ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA.

Techniques: Expressing, ChIP-sequencing

Journal: bioRxiv

Article Title: KDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity

doi: 10.1101/561571

Figure Lengend Snippet: (A) A schematic representation of the Kdm2a/b-CxxC fl/fl mESC line, in which loxP sites were inserted into the Kdm2a and Kdm2b genes flanking exons that encode the ZF-CxxC domain. sgRNA.1 and sgRNA.2 indicate the position of CRISPR-mediated loxP insertion. (B) ChIP-qPCR analysis showing KDM2A (left panel) and KDM2B (right panel) enrichment relative to input in Kdm2a/b-CXXC fl/fl mESCs before (UNT) and after tamoxifen treatment (OHT). Error bars show standard error of the mean of four biological replicates. (C) Digital droplet PCR analysis, showing the fold change in template cDNA concentration following tamoxifen treatment (OHT) of Kdm2a/b-CXXC fl/fl mESCs (blue). Error bars show standard deviation of three biological replicates. The fold change calculated by cnRNA-seq is shown for comparison (green), and the significance of this change is annotated below the graph. The dashed lines represent no change in expression (black) and the 1.4 fold change threshold used for cnRNA-seq analysis (red). (D) An MA-plot showing log2 fold change in gene expression in Kdm2a/b-CXXC fl/fl mESCs following tamoxifen treatment (OHT), normalising nRNA-seq data to total library size. The number of genes with significantly increased or decreased expression (p-adj <0.05 and > 1.4-fold) is shown in red and density of gene expression changes is shown on the right.

Article Snippet: Chromatin was pre-cleared for 1 hour with either protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) or protein A agarose beads (Repligen, for histone or RNAPII ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA.

Techniques: CRISPR, Concentration Assay, Standard Deviation, Expressing

Journal: bioRxiv

Article Title: KDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity

doi: 10.1101/561571

Figure Lengend Snippet: (A) A schematic of the Kdm2a/b-CXXC fl/fl system in which addition of tamoxifen (OHT) leads to the generation of KDM2 proteins that lack the ZF-CxxC domain and therefore are unable to bind to chromatin. (B) Western blot analysis for KDM2A and KDM2B in Kdm2a/b-CXXC fl/fl mESCs before (UNT) and after 96 hours tamoxifen (OHT) treatment. BRG1 is shown as a loading control for both blots. Asterisks indicate non-specific bands. (C) Genomic snapshots showing gene expression (cnRNA-seq) before (UNT) and after tamoxifen (OHT) treatment of Kdm2a/b-CXXCfl/fl mESCs, for representative genes that moderately ( Fbxl20 , left) or more dramatically increased in expression ( Fosl2 , right). BioCAP and KDM2A and KDM2B ChIP-seq signal are shown for reference ( ; ; ) (D) An MA-plot showing log2 fold change in gene expression (cnRNA-seq) in K dm2a/b-CXXC fl/fl mESCs following tamoxifen treatment. The number of genes with significantly increased or decreased expression (p-adj < 0.05 and > 1.4-fold) are shown in red and density of gene expression changes is shown on the right. (E) A density plot showing the distribution of the log2 fold change in gene expression following tamoxifen treatment of Kdm2a/b-CXXC fl/fl mESCs, for all genes. (F) A bar graph showing the proportion of genes that have a CGI promoter, for all genes and genes that significantly increased or decreased in expression following tamoxifen treatment of Kdm2a/b-CXXC fl/fl mESCs.

Article Snippet: Chromatin was pre-cleared for 1 hour with either protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) or protein A agarose beads (Repligen, for histone or RNAPII ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA.

Techniques: Western Blot, Expressing, ChIP-sequencing

Journal: bioRxiv

Article Title: KDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity

doi: 10.1101/561571

Figure Lengend Snippet: (A) Metaplots showing enrichment of BioCAP signal and KDM2A, KDM2B, RING1B and SUZ12 ChIP-seq signal at the TSS of all CGI-associated genes (n=14106, green) and of the subset of these genes that significantly increased in expression following tamoxifen treatment of Kdm2a/b-CXXC fl/fl mESCs (n=3879, red) ( ; ; ). (B) Left: an MA-plot showing log2 fold change in gene expression (cnRNA-seq) in Pcgf1 fl/fl mESCs following tamoxifen treatment to induce PCGF1 knockout . The number of genes with significantly increased or decreased expression (p-adj < 0.05 and > 1.4-fold) is shown in red and density of gene expression changes is shown on the right. Right: as (B) but for Kdm2a/b-CXXC fl/fl mESCs, shown for comparison. (C) A bar graph comparing the distribution of genes into three classes – non-CGI, polycomb (PRC) occupied and non-PRC occupied – for all genes and for genes that that significantly increased in expression following tamoxifen treatment of Pcgf1 fl/ fl or Kdm2a/b-CXXC fl/fl mESCs. Non-CGI genes are genes that lack a CGI at their promoter. Non-PRC-occupied genes have a CGI promoter that is not bound by polycomb complexes, while PRC-occupied genes have a CGI promoter that is bound by polycomb complexes. (D) A Venn diagram showing the overlap between genes that significantly increased in expression following tamoxifen treatment of Pcgf1 fl/ fl and Kdm2a/b-CXXC fl/fl mESCs. (E) A box plot showing the starting expression level (log2 UNT RPKM) for genes grouped into deciles based on their log2 fold change in expression following tamoxifen treatment of Kdm2a/b-CXXC fl/fl mESCs. (F) Gene ontology analysis of genes that significantly increased in expression following tamoxifen treatment of Kdm2a/b-CXXC fl/fl mESCs. (G) As (H), but for the subset of significantly increasing genes that were not classified as polycomb target genes.

Article Snippet: Chromatin was pre-cleared for 1 hour with either protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) or protein A agarose beads (Repligen, for histone or RNAPII ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA.

Techniques: ChIP-sequencing, Expressing, Knock-Out

Journal: bioRxiv

Article Title: KDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity

doi: 10.1101/561571

Figure Lengend Snippet: (A) A scatter plot comparing the log2 fold change in gene expression (cnRNA-seq) following tamoxifen treatment of Kdm2a/b-CXXC fl/fl and Pcgf1 fl/fl mESCs. The solid line shows the linear regression, and the coefficient of determination (R 2 ) and Spearman correlation coefficient (Cor) are annotated.

Article Snippet: Chromatin was pre-cleared for 1 hour with either protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) or protein A agarose beads (Repligen, for histone or RNAPII ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA.

Techniques: Expressing

Journal: bioRxiv

Article Title: KDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity

doi: 10.1101/561571

Figure Lengend Snippet: (A) A schematic of the Kdm2a-CXXC fl/fl and Kdm2b-CXXC fl/fl systems in which addition of tamoxifen (OHT) leads to the generation of KDM2A or KDM2B proteins that lack the ZF-CxxC domain, respectively, and therefore are unable to bind to chromatin. (B) MA-plots showing log2 fold change in gene expression (cnRNA-seq) in Kdm2a-CXXC fl/fl (left) or Kdm2b-CXXC fl/fl mESCs (right) following tamoxifen treatment. The number of genes with significantly increased or decreased expression (p-adj < 0.05 and > 1.4-fold) are shown in red and density of gene expression changes is shown on the right. (C) A density plot showing the distribution of the log2 fold change in gene expression following tamoxifen treatment of Kdm2a-CXXC fl/fl , Kdm2b-CXXC fl/fl or Kdm2a/b-CXXC fl/fl mESCs, for all genes. (D) A bar graph illustrating the distribution of genes between three gene classes (Non-CGI, Non-PRC, PRC) described in , for all genes and for genes that significantly increased or decreased in expression following tamoxifen treatment of Kdm2b-CXXC fl/fl mESCs. (E) Gene ontology analysis of genes that significantly increased in expression following tamoxifen treatment of Kdm2b-CXXC fl/fl mESCs. (F) As (E), but for the subset of significantly increasing genes that were not classified as polycomb target genes. (G) A scatter plot comparing the log2 fold change in gene expression (cnRNA-seq) following tamoxifen treatment of Kdm2b-CXXC fl/fl and Kdm2a/b-CXXC fl/fl mESCs. The solid line shows the linear regression, and the coefficient of determination (R 2 ) and Spearman correlation coefficient (Cor) are annotated. (H) A scatter plot of the log2 fold change in gene expression (cnRNA-seq) following tamoxifen treatment of Kdm2a/b-CXXC fl/fl mESCs for genes that significantly increased in expression, plotted against the ratio of the log2 fold change in gene expression following tamoxifen treatment of Kdm2b-CXXC fl/fl versus Kdm2a/b-CXXC fl/fl mESCs. A 1.5-fold threshold (red dotted lines) was used to define genes which were differentially regulated between the two datasets, and the number of genes with more than 1.5-fold increased or decreased expression is shown in red. The solid line shows the linear regression, and the coefficient of determination (R 2 ) and Spearman correlation coefficient (Cor) are annotated.

Article Snippet: Chromatin was pre-cleared for 1 hour with either protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) or protein A agarose beads (Repligen, for histone or RNAPII ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA.

Techniques: Expressing

Journal: bioRxiv

Article Title: KDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity

doi: 10.1101/561571

Figure Lengend Snippet: (A) Western blot analysis for KDM2A and KDM2B in K dm2a-CXXC fl/fl mESCs before (UNT) and after 96 hours of tamoxifen treatment (OHT). BRG1 is shown as a loading control for both blots. Asterisks indicate non-specific bands. (B) As (A) but for K dm2b-CXXC fl/fl mESCs.

Article Snippet: Chromatin was pre-cleared for 1 hour with either protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) or protein A agarose beads (Repligen, for histone or RNAPII ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA.

Techniques: Western Blot

Journal: bioRxiv

Article Title: KDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity

doi: 10.1101/561571

Figure Lengend Snippet: (A) An MA-plot showing log2 fold change in the accessibility (cATAC-seq) of CGI-associated gene promoters in K dm2a/b-CXXC fl/fl mESCs following tamoxifen treatment. No promoters significantly changed in accessibility (p-adj < 0.05 and > 1.4-fold). (B) A heatmap of the fold change in RNAPII ChIP-seq signal following tamoxifen treatment of K dm2a/b-CXXC fl/fl mESCs, for CGI-associated (n=14106) and non-CGI-associated (n=6527) gene promoters. (C) A metaplot showing RNAPII enrichment at CGI-associated genes before (UNT) and after tamoxifen treatment (OHT) Kdm2a/b-CXXC fl/fl mESCs. (D) Genomic snapshots showing RNAPII occupancy before (UNT) and after tamoxifen treatment (OHT) of Kdm2a/b-CXXC fl/fl mESCs, for (above) a representative gene that retains RNAPII at the promoter and increases in RNAPII occupancy throughout the gene body, and (below) a representative gene that decreases in RNAPII at both promoter and gene body regions. BioCAP and KDM2A and KDM2B ChIP-seq signal are shown for reference ( ; ; ). (E) Empirical cumulative density function (ECDF) plots of RNAPII pausing index for CGI-associated (left) or non-CGI-associated (right) genes, before (UNT) and after tamoxifen treatment (OHT) of Kdm2a/b-CXXC fl/fl mESCs. (F) Metaplots showing Ser5P-RNAPII (upper panel) or Ser2P-RNAPII (lower panel) enrichment at CGI-associated genes before (UNT) and after tamoxifen treatment (OHT) of Kdm2a/b-CXXC fl/fl mESCs. (G) A boxplot showing the fold change in Ser5P-RNAPII at gene promoters (left) or Ser2P-RNAPII at gene bodies (right) following tamoxifen treatment of Kdm2a/b-CXXC fl/fl mESCs, normalised to RNAPII-NTD signal. The fold changes for CGI-associated and non-CGI-associated genes are shown.

Article Snippet: Chromatin was pre-cleared for 1 hour with either protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) or protein A agarose beads (Repligen, for histone or RNAPII ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA.

Techniques: ChIP-sequencing

Journal: bioRxiv

Article Title: KDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity

doi: 10.1101/561571

Figure Lengend Snippet: (A) MA-plots showing log2 fold change in the accessibility (cATAC-seq) of CGI-associated gene promoters in K dm2a-CXXC fl/fl (left) or K dm2a-CXXC fl/fl (right) mESCs following tamoxifen treatment. No promoters significantly changed in accessibility (p-adj < 0.05 and > 1.4-fold). (B) An illustration of the pausing index, the ratio of the average read density of RNAPII-NTD at the promoter and the average read density of RNAPII in the gene body. (C) Scatter plots comparing the log2 fold change in gene expression (cnRNA-seq) with the log2 fold change in RNAPII occupancy (ChIP-seq) following tamoxifen treatment of Kdm2a/b-CXXC fl/fl mESCs, at CGI-associated gene promoters (left) or gene bodies (right). The solid line shows the linear regression, and the coefficient of determination (R 2 ) and Spearman correlation coefficient (Cor) are annotated. (D) Boxplots showing the fold change in RNAPII occupancy at CGI-associated gene promoters (left) or gene bodies (right) following tamoxifen treatment of Kdm2a/b-CXXC fl/fl mESCs, for all CGI genes or for genes that significantly increased in expression separated into quartiles according to their log2 fold change in expression (Q1 < Q2 < Q3 < Q4). (E) A metaplot showing RNAPII enrichment before (UNT) and after tamoxifen treatment (OHT) of Kdm2a/b-CXXC fl/fl mESCs, for the top quartile of significantly upregulated genes (Q4 – as in (D)). (F) Heatmap analyses of the fold change in RNAPII, Ser5-RNAPII or Ser2P-RNAPII ChIP-seq signal following tamoxifen treatment of K dm2a/b-CXXC fl/fl mESCs, for CGI-associated (n=14106) and non-CGI-associated (n=6527) gene promoters.

Article Snippet: Chromatin was pre-cleared for 1 hour with either protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) or protein A agarose beads (Repligen, for histone or RNAPII ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA.

Techniques: Expressing, ChIP-sequencing