Macrophage differentiation is marked by increased abundance of the mRNA 3’ end processing machinery, altered poly(A) site usage, and sensitivity to the level of CstF64

Regulation of mRNA polyadenylation is important for response to external signals and differentiation in several cell types, and results in mRNA isoforms that vary in the amount of coding sequence or 3’ UTR regulatory elements. However, its role in differentiation of monocytes to macrophages has not been investigated. Macrophages are key effectors of the innate immune system that help control infection and promote tissue-repair. However, overactivity of macrophages contributes to pathogenesis of many diseases. In this study, we show that macrophage differentiation is characterized by shortening and lengthening of mRNAs in relevant cellular pathways. The cleavage/polyadenylation (C/P) proteins increase during differentiation, suggesting a possible mechanism for the observed changes in poly(A) site usage. This was surprising since higher C/P protein levels correlate with higher proliferation rates in other systems, but monocytes stop dividing after induction of differentiation. Depletion of CstF64, a C/P protein and known regulator of polyadenylation efficiency, delayed macrophage marker expression, cell cycle exit, attachment, and acquisition of structural complexity, and impeded shortening of mRNAs with functions relevant to macrophage biology. Conversely, CstF64 overexpression increased use of promoter-proximal poly(A) sites and caused the appearance of differentiated phenotypes in the absence of induction. Our findings indicate that regulation of polyadenylation plays an important role in macrophage differentiation.


Supplementary Figures
Supplementary Figure S1. Morphological changes and altered expression of cell-surface markers and mRNAs during differentiation of THP-1 cells and human primary monocytes.
(A) The effect of differentiation on THP-1 cellular morphology. THP-1 cells exposed to PMA (30 nM) for 6 and 24 hours and observed by phase contrast microscopy (40X). (B) Western blots of total cell lysates from THP-1 cells differentiated with PMA (30 nM) for 0, 1, 6, 18 and 24 hours. Blots were probed with antibodies against CD16, CD68, HLA-DRA, ICAM1, CD38 and CD14 with βactin as the loading control. (C) Attachment assay for THP-1 cells, performed as described for Fig.  1C. (D) Proliferation assay for THP-1 cells performed as described for Fig. 1D. (E) Effect of THP-1 differentiation on the expression level of mRNAs from genes involved in macrophage differentiation and function. RT-qPCR assays performed for genes as described in Fig. 1E. The figure represents mean ± SE from three independent experiments. P value <0.05 was considered significant, where * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001.

Supplementary Figure S2. Gene set enrichment analysis of mRNAs with expression changes in U937 cells after PMA treatment.
Functional annotation clustering of mRNAs whose expression is regulated after 6h (A) and 24h (B) of differentiation. The 10 most significant GO-process enriched gene groups according to GSEA analysis are ranked based on negative log10 (P-values) for upregulated (left panel) and downregulated (right panel) transcripts. Green indicates biological process; red, canonical process. Pathways classified according to specific cellular processes are grouped together as indicated by the individual symbols at the bottom of the graphs.

Supplementary Figure S3. Changes in poly(A) site use in U937 cells after 6 hours of PMA treatment and validation by 3'RACE.
(A) UCSC genome browser plots of RNA sequencing tracks highlighting the 3′-UTR profile differences for shortened and lengthened genes after 6h PMA treatment with respect to control (0h). The colors of the tracks represent 0h (red) and 6h (blue). Proximal (P) and distal (D) poly(A) sites are indicated with red stars. The green arrow defines the direction of the coding strand, blue arrow defines the direction of chromosome co-ordinates and tag counts are indicated on the y axis. Additionally, positions and chromosome co-ordinates of the annotated PACs are indicated at the top of each browser plot. (B) Quantitative bar graphs reflecting the differences in APA for shortened and lengthened targets. The mean relative usage of proximal poly(A) site with respect to the total read counts at the proximal and distal poly(A) sites as visualized in the UCSC genome browser is plotted for each target. Unpaired t-test was performed to determine the significance between the treatment groups and P value <0.05 was considered significant where * = P ≤ 0.05. (C) Representative 1% agarose gel images for the 3′ RACE RT-PCR from U937 cells (untreated and 6h PMA-treated) for shortened gene CIAPIN1 and lengthened gene EIF1 (upper panel) using gene specific primer at the last exon-exon junction and reverse anchor primer. Normalized intensities of long and short bands for each PCR were quantified and a long/short ratio determined and normalized to the No PMA control. The graph (lower panel) represents mean ± SE from at least two independent experiments. P value <0.05 was considered significant, where * = P ≤ 0.05; ** = P ≤ 0.01. Figure S4. Validation of APA events in THP-1 and human primary monocytes by 3' RACE.

Supplementary
Representative 1% agarose gel images for the 3′ RACE RT-PCR from control and differentiated THP-1 cells and human primary monocytes for shortened genes SERPINB1 and PTCH1 (A and C), lengthened genes PSAT1 and PSMD10 (B and D) with gene specific primer at the last exon-exon junction and reverse anchor primer. Normalized intensities of long and short bands for each PCR were quantified and a long/short ratio determined and normalized to the No PMA or undifferentiated controls. The corresponding graphs (right) represents mean ± SE from at least two independent experiments. P value <0.05 was considered significant, where * = P ≤ 0.05; ** = P ≤ 0.01.