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. 2022 Jul 1;4(3):103-115.
doi: 10.1097/BS9.0000000000000122. eCollection 2022 Jul.

KHSRP combines transcriptional and posttranscriptional mechanisms to regulate monocytic differentiation

Jiayue Xu  1   2 , Dongsheng Wang  3 , Hongliu Ma  4 , Xueying Zhai  1   2 , Yue Huo  1   2 , Yue Ren  1   2 , Weiqian Li  1   2 , Le Chang  1   2 , Dongxu Lu  1   2 , Yuehong Guo  1   2 , Yanmin Si  1   2 , Yufeng Gao  1   2 , Xiaoshuang Wang  1   2 , Yanni Ma  1   2 , Fang Wang  1   2 , Jia Yu  1   2   5
Affiliations

KHSRP combines transcriptional and posttranscriptional mechanisms to regulate monocytic differentiation

Jiayue Xu et al. Blood Sci. .

Abstract

RNA-binding proteins (RBPs) are widely involved in the transcriptional and posttranscriptional regulation of multiple biological processes. The transcriptional regulatory ability of RBPs was indicated by the identification of chromatin-enriched RBPs (Che-RBPs). One of these proteins, KH-type splicing regulatory protein (KHSRP), is a multifunctional RBP that has been implicated in mRNA decay, alternative splicing, and miRNA biogenesis and plays an essential role in myeloid differentiation by facilitating the maturation of miR-129. In this study, we revealed that KHSRP regulates monocytic differentiation by regulating gene transcription and RNA splicing. KHSRP-occupied specific genomic sites in promoter and enhancer regions to regulate the expression of several hematopoietic genes through transcriptional activation and bound to pre-mRNA intronic regions to modulate alternative splicing during monocytic differentiation. Of note, KHSRP had co-regulatory effects at both the transcriptional and posttranscriptional levels on MOGOH and ADARB1. Taken together, our analyses revealed the dual DNA- and RNA-binding activities of KHSRP and have provided a paradigm to guide the analysis of other functional Che-RBPs in different biological systems.

Keywords: KHSRP; Monocytic differentiation; RNA-binding proteins; Transcriptional regulation.

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Conflict of interest statement

The authors declare that they have no conflicts of interest related to the subject matter or materials discussed in this article.

Figures

Figure 1.
Figure 1.
Identification of splicing regulatory patterns by integration of CLIP-seq and RNA-seq datasets (A) Plot indicating the number of AS events (upper panel). Identification of 4 major AS patterns by comparison of shKHSRP and Ctrl RNA-seq data; skipped exon (SE, red), alternative 3’ splice site (A3SS, orange), alternative 5’ splice site (A5SS, green), and retained intron (RI, blue). (B) Density of KHSRP eCLIP region-level enrichment in 3’-UTR, CDS, intron, and exon regions. (C) Pie chart showing the proportion of protein-coding genes (orange) and nonprotein coding genes (yellow), including lncRNA (purple), pseudo gene (pink), scaRNA (light blue), snRNA (dark blue), and TEC (light green), that were associated with KHSRP. (D) Venn diagram showing the region of intersection of AS events, including those that were absent or present following KHSRP knockdown and the eCLIP enriched region. (E) Enrichment network representing the top-12 enriched terms for AS events located in eCLIP enriched region. (F) Plot showing AS events following KHSRP knockdown (x-axis) vs (y-axis) fold enrichment in KHSRP eCLIP for the indicated transcript-binding regions. (G) Distribution of ΔΨ changes following KHSRP knockdown in the eCLIP enrichment region (P < .05 indicates statistical significance by Wilcoxon rank-sum test). (H) Sashimi plots showing differential AS events in shKHSRP (dark green) and control (light green) samples (upper panel). Alternatively, spliced exons are shown in green. Bars indicate eCLIP signals on gene loci in the IP (yellow) and input (orange) (lower panel).
Figure 2.
Figure 2.
Integration of ChIP-seq and RNA-seq data identifies KHSRPs that interact with chromatin and activate gene transcription. (A, B) Volcano plot (A) and heatmap (B) showing differentially expressed genes between shKHSRP and control samples. (C, D) GO functional enrichment analysis of activated (C) and repressed genes (D) in shKHSRP sample compared with control sample. (E) Percentages of different genic and intergenic regions associated with KHSRP peaks identified by ChIP-seq in THP-1 cells (KHSRP-bound). The abundance of each type of region in the human genome (Genome) is shown for comparison. The numbers in parentheses indicate the enrichment ratio relative to the genome; P values were determined by single-tailed Fisher’s exact test (*P < .05). (F) Metaplot showing the distribution of H3K4me1 and H3K27ac ChIP-seq (left panel) and Pol II ChIP-seq fragment depth (right panel) within the −3000 bp to +3000 bp region around the KHSRP ChIP-seq peaks in the enhancer and promoter regions of 168 transcriptionally activated genes. (G) Venn diagram illustrating the intersection of KHSRP-bound genes and KHSRP-activated genes. (H) Percentages of different genic and intergenic regions associated with KHSRP peaks as identified by ChIP-seq in 168 KHSRP transcriptionally activated genes. (I) Metaplot showing the distribution of H3K4me1 and H3K27ac ChIP-seq (left panel) and Pol II ChIP-seq fragment depths (right panel) within the region −3000 bp to +3000 bp around the KHSRP ChIP-seq peaks in the enhancer and promoter regions of 168 transcriptionally activated genes. (J) Enrichment network representing the top 10 enriched terms of 168 KHSRP transcriptionally activated genes. (K) Genomic visualization of KHSRP, H3K4me1, K3H27ac, and Pol II ChIP-seq datasets at the indicated gene loci. (L) Nearby TF-binding motif prediction by SpaMo was used to identify putative hematopoiesis-related interaction partner TFs for KHSRP. The top-5 identified TFs determined by e-value are shown in the figure. The number under the x-axis represents the best gap between the KHSRP motif and indicated TF motifs.
Figure 3.
Figure 3.
Comparative analysis of transcriptional and posttranscriptional functions (A) Number of differentially expressed genes without AS events between shKHSRP and control samples (DEG-only group), genes with AS events without DEG between shKHSRP and control samples (AS-only group), and differentially expressed genes with AS events between shKHSRP and control samples (Both group). (B) Heatmap displaying expression level of 192 Both group genes (left) and difference inclusion levels (DILs) of relative AS events (middle). Bar plot showing enriched terms in the 4 groups (right) that were downregulated in DEG and upregulated in DIL (pink), downregulated in DEG and downregulated in DIL (blue), upregulated in DEG and upregulated in DIL (orange), or downregulated in DEG and upregulated in DIL (green). (C) Comparison of occupied gene numbers (left) and peak numbers (right) between ChIP-seq and CLIP-seq datasets of the 4 groups: all enrichment genes on the genome (Total), DEG-only group, AS-only group, and Both group. (D) ChIP-seq signal distribution (left) and CLIP-seq signal distribution (right) with human genomic intrinsic constitution in a comparison of the 4 groups (Total, DEG-only, AS-only, and Both). (E) Heatmap presenting the occupation ratio of histone marker ChIP signals colocalized with ChIP peaks at promoter and gene body regions of co-occupied genes in the 4 groups (Total, DEG-only, AS-only, and Both). (F) Comparison of co-occupied genes from ChIP-seq and CLIP-seq datasets of the 4 groups (Total, DEG-only, AS-only, and Both). The x-axis shows the Jaccard index of the ChIP-seq and CLIP-seq occupied genes of each group; bubble size indicates co-occupied gene number. (G) Circos plot describing the overall multi-layered relationship of whole genomes; bars represent the DIL of AS events (DIL > 0, orange; DIL
Figure 4.
Figure 4.
KHSRP regulates alternative splicing during monocyte differentiation (A) Pie chart showing the proportion of major patterns of AS events following PMA treatment (left panel) and proportion of eCLIP enriched regions. (B) 3D scatterplot presenting the relationship among AS events following PMA treatment and following KHSRP and eCLIP enrichment. (C) GO functional enrichment analysis of AS events following PMA treatment and KHSRP knockdown and PMA treatment combined with KHSRP knockdown with eCLIP peaks. (D) Sashimi plots showing differential AS events both in the shKHSRP sample (red) and control sample (pink) and in PMA-treated samples (PMA 0 h and 48 h shown in light and dark blue, respectively). Alternatively, spliced exons are shown in orange. (E) Sashimi plots showing differential AS events both in shKHSRP- and PMA-treated samples (upper panel); bars indicate eCLIP signals on gene loci in IP (green) and input (orange) (lower panel).
Figure 5.
Figure 5.
KHSRP participates in the regulation of monocyte differentiation at the transcriptional level (A) Scatterplot showing comparison of DEGs between the shKHSRP and control samples and the PMA-treated and control samples. (B) GO functional enrichment analysis of DEGs commonly affected by KHSRP and PMA. (C) Venn diagram showing common DEGs between the shKHSRP and control samples, DEGs between the PMA-treated and control samples, and genes with KHSRP peaks identified by ChIP-seq. (D) Circos plot showing genes and GO enrichment of DEGs affected by both shKHSRP and PMA, with enrichment peaks identified by ChIP-seq. (E) Genomic visualization of KHSRP, H3K27ac, H3K4me1, and Pol II ChIP-seq datasets for the indicated gene loci (left panel), and bar plot showing expression levels in the shKHSRP group compared with the control group and the PMA group compared with the control group.
Figure 6.
Figure 6.
Characterization of potential co-transcriptional genes in monocytic differentiation. (A) Venn diagram showing the intersection of transcriptional regulatory genes and posttranscriptional regulatory genes in monocytic differentiation. (B, C) Sashimi plots showing differential AS events in both shKHSRP- and PMA-treated samples (left panel), and bar plot showing the expression levels of DEGs and genomic visualization of KHSRP, H3K27ac, H3K4me1, and Pol II ChIP-seq datasets and eCLIP signal on the indicated gene loci (right panel). (D) Schematic diagram of KHSRP’s transcriptional and posttranscriptional regulatory function during monocytic differentiation.
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