Fig 2: Absolute per Cell-Normalized Chromatin Immunoprecipitation Reveals Global Effects of HMGN1 Overexpression on Histone Marks(A) Metagene plots and read quantitation of H3K27ac and H3K4me3 ChIP-seq in Nalm6 cells stably expressing a doxycycline-inducible HMGN1 cDNA 6 hours after addition of doxycycline or vehicle to the culture medium. Plots on the left are relative median read count-normalized “traditional” ChIP-seq, and on the right, absolute per cell-normalized to sequencing reads from ChIP-seq of exogenous spiked-in Drosophila S2 cell nuclei. RPM, reads per million; nRPM, normalized reads per million; x axis of metagene plots shows the transcriptional start site (TSS) and 5 kb upstream and downstream. Boxplots represent the distribution of ChIP-seq peak height at each detected gene (compared by t test).(B) Gene tracks showing a representative locus on chr17 where an increase in H3K27 acetylation after addition ofdoxycycline is only evident after applying per cell absolute spike-in normalization.(C) Relative read counts of histone marks at peaks within 1 kb of the TSS of genes that significantly (fold-change >1.5, p < 0.05) increased (UP, red) or decreased (DOWN, blue) in expression after induction of HMGN1 overexpression. Distributions compared by t test.

Fig 3: HMGN1 Increases RNA Content after Short-Term Induction, Requires Residues Necessary for Nucleosome Binding, and Induces Greater Nascent Transcription(A) Western blotting of lysates from Nalm6 B-ALL cells stably infected with a lentivirus that contains a doxycycline-inducible human HMGN1 cDNA (N1), an HMGN1 mutant that lacks the ability to bind to the nucleosome, HMGN1-S20,24E (SE), or empty vector (V) at baseline (0 hr), 3 hr, and 6 hr after the addition of doxycycline to the culture medium.(B) Total RNA content measured by fluorometric assay in biological triplicates of the indicated cells relative to empty vector over a time course after addition of doxycycline to the culture medium. n = 3 biological replicates. Samples compared by t test.(C) Left: expression ratios across all expressed genes from RNA-sequencing of Nalm6 cells 6 hr after addition of doxycycline to overexpress HMGN1 or HMGN1-SE, calculated using relative or absolute spike-in normalization (n = 4 biological replicates per condition). Right: dot plot of individual gene expression comparing HMGN1 to HMGN1-SE expressing Nalm6 cells after doxycycline induction using relative or absolute normalization. The dotted line represents the unity line of no difference between conditions. The contour lines and legend represent high (green) to low (blue) relative bin density. Red and blue numbers represent the number of genes that increase or decrease, respectively, in HMGN1-WT versus HMGN1-SE (fold-change >1.5, p < 0.05). FPKM, fragments per kilobase of transcript per million mapped reads; nFPKM, ERCC-normalized FPKM.(D) Distribution of fold change in individual gene expression between HMGN1 and HMGN1-SE overexpressing cells from relative (orange) or absolute normalized (red) RNA-seq analysis. Positive fold change = higher in HMGN1 compared to HMGN1-SE. Plots are divided into low, mid, and high terciles by gene expression in HMGN1-SE cells, similarly to Figure 2A. Distributions compared by t test.(E) Cumulative distribution function plot of SLAM-seq data for active genes showing the log2 fold change in nascent reads during the labeling period in induced compared to uninduced cells. The red line represents cells after induction of HMGN1 and the gray line represents cells after induction of the HMGN1-SE mutant (n = 3 biological replicates). The rightward shift of the curves suggests that per gene there are more transcripts generated after HMGN1 induction compared to uninduced cells or to cells expressing the HMGN1-SE mutant. Changes in distributions are significant at p < 1e–9 by two-tailed t test.(F) Binned plot of active genes ranked by nascent transcript fraction in uninduced cells (x axis) versus the mean log2 fold change in the conversion ratio (number of nascent transcripts generated in the labeling period relative to pre-existing transcripts) for genes in a bin, in induced or uninduced cells (y axis). Error bars represent 95% confidence intervals of the mean.(G) Conversion fraction (nascent transcripts per total transcripts) of an illustrative gene, PRDM1, in uninduced cells, cells after induction of HMGN1, or cells after induction of the HMGN1-SE mutant. Error bars represent SEM.

Fig 4: Model for Gene Expression Changes Associated with Trisomy 21 or HMGN1 OverexpressionAbsolute per cell normalization of transcriptome data may provide a mechanistic explanation for some previous, incompletely explained observations in trisomy 21 cells, including and global RNA and epigenomic changes. If the actual expression pattern in trisomy 21 (or with HMGN1 overexpression) is amplification that differentially affects areas of baseline “high” or “low” expression (left), then forced normalization to an equivalent median signal between samples (blue arrow and dotted line) would instead be interpreted as decreased expression in baseline “high” expressed genes and increased expression in “low” expressed genes (right).

Fig 5: Transcriptome Changes in Ts1Rhr and HMGN1-Overexpressing B Cells Are Widely Distributed but Enriched at Genes Associated with Certain Histone Modifications(A) Distribution of fold change in individual gene expression between Ts1Rhr (top) or HMGN1-OE (bottom) and wild-type B cell progenitors from relative (orange) or cell count-normalized (“Absolute,” red) RNA-seq analysis. Positive fold change = higher in Ts1Rhr or HMGN1 compared to WT. Plots are all expressed genes (FPKM >0.1) divided into low, mid, and high terciles of equal size by gene expression in wild-type cells. Distributions compared by t test.(B) Median H3K4m3 and H3K27me3 histone marks in wild-type B cell progenitors, plotted as median mark intensity in each of 50 bins of genes ranked by expression in wild-type cells, as in Figure 1C.(C) Left: gene set enrichment analysis of all expressed genes in HMGN1 overexpressing compared to wild-type B cell progenitors of polycomb repressor complex 2 and H3K27-related datasets in the C2 CGP collection of MSigDB (Broad Institute) shows significant enrichment of EZH2 target genes in hematopoietic stem cells (KAMMINGA_EZH2_TARGETS) and genes marked by H3K27me3 in reprogrammed iPS cells (MIKKELSEN_MCV6_HCP_WITH_H3K27ME3). ES, enrichment score; FDR, false discovery rate (adjusted p value). Right: heatmap of expression level of the “leading edge” genes in the Kamminga gene set from GSEA analysis in biological triplicate samples of wild-type, Ts1Rhr, and HMGN1-OE B cell progenitors (blue, low; red, high expression).