Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jan 23;43(1):113662.
doi: 10.1016/j.celrep.2023.113662. Epub 2024 Jan 6.

TDP-43 chronic deficiency leads to dysregulation of transposable elements and gene expression by affecting R-loop and 5hmC crosstalk

Yingzi Hou  1 , Yangping Li  1 , Jian-Feng Xiang  1 , Kedamawit Tilahun  2 , Jie Jiang  2 , Victor G Corces  1 , Bing Yao  3
Affiliations

TDP-43 chronic deficiency leads to dysregulation of transposable elements and gene expression by affecting R-loop and 5hmC crosstalk

Yingzi Hou et al. Cell Rep. .

Abstract

TDP-43 is an RNA/DNA-binding protein that forms aggregates in various brain disorders. TDP-43 engages in many aspects of RNA metabolism, but its molecular roles in regulating genes and transposable elements (TEs) have not been extensively explored. Chronic TDP-43 knockdown impairs cell proliferation and cellular responses to DNA damage. At the molecular level, TDP-43 chronic deficiency affects gene expression either locally or distally by concomitantly altering the crosstalk between R-loops and 5-hydroxymethylcytosine (5hmC) in gene bodies and long-range enhancer/promoter interactions. Furthermore, TDP-43 knockdown induces substantial disease-relevant TE activation by influencing their R-loop and 5hmC homeostasis in a locus-specific manner. Together, our findings highlight the genomic roles of TDP-43 in modulating R-loop-5hmC coordination in coding genes, distal regulatory elements, and TEs, presenting a general and broad molecular mechanism underlying the contributions of proteinopathies to the etiology of neurodegenerative disorders.

Keywords: CP: Neuroscience; DNA modification; R-loops; TDP-43; neurodegeneration; transposable element.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. TDP-43 knockdown results in slowed cell proliferation and impaired response to DNA damage
(A) qRT-PCR analysis of TDP-43 mRNA levels in shNC and shTDP-43 cells (n = 3, Student’s t test). (B) Western blot analysis of TDP-43 protein levels in shNC and shTDP-43 cells. (C) Quantification of TDP-43 protein levels normalized to α-tubulin (n = 3, Student’s t test). (D) Representative immunofluorescence images of TDP-43 cellular expression. Scale bar: 20 μm. (E) Quantification of immunofluorescence images. The graph shows the median intensity of TDP-43, and around 150 cells were counted. The p value was calculated by Student’s t test. (F) Growth curve of shNC and shTDP-43 cells using XTT assay. The p value was calculated by two-way ANOVA with the Geisser-Greenhouse correction. (G) Measurement of caspase-3/7 activity indicated by luminescence intensity (n = 12, Student’s t test). Error bars represent SEM. n represents number of replicates. **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 2.
Figure 2.. Genome-wide R-loops are dysregulated in the TDP-43 stable knockdown cell line
(A) Proportions of gene body, intergenic region, and promoter annotated by all R-loop peaks identified in shNC cells. Gene body, from the transcription start site (TSS) to the transcription termination site (TTS); promoter, from −1 kb upstream to +100 bp downstream of the TSS. (B) Volcano plot of the DRIP-seq results. The red dots indicate significantly accumulated R-loops, and the blue dots indicate significantly depleted R-loops upon TDP-43 knockdown (DESeq2, n = 3, FDR < 0.05). (C) ngs.plot analysis of R-loop reads over the 1,535 R-loop-gained regions. (D) ngs.plot analysis of R-loop reads over the 1,743 R-loop-lost regions. (E) Enrichment analysis of the 1,535 significantly upregulated R-loops (red) and the 1,743 significantly downregulated R-loops (blue) in different genomic features. The comparison of differential R-loop peak numbers in various gene features with expected R-loop peak numbers based on the percentage of the length of each chromatin feature is shown as log2 observed/expected (log2 Obs/Exp). (F) Visual analysis of R-loops in the gene bodies of NPAS4 (left) and NECTIN2 (right) using the Integrative Genomic Viewer (IGV) browser. (G) DRIP-qPCR analysis of R-loops in NPAS4 (left) and NECTIN2 (right). P1, primer pair 1 in (F); P2, primer pair 2 in (F). RNase H-treated samples are negative controls. Data are a summary of three replicates. Error bars represent SEM. All the p values were calculated by Student’s t test. *p < 0.05; **p < 0.01; ****p < 0.0001.
Figure 3.
Figure 3.. The crosstalk between TDP-43-mediated R-loops and 5hmC regulates a group of genes
(A) Scatterplot of the RNA-seq results (Cuffdiff, n = 3, FDR < 0.05). Average CPM, average read counts per million across all three replicates. (B) ngs.plot analysis of R-loop reads over the 2,030 downregulated genes in (A). The p value was calculated by Student’s t test. (C) Heatmap of R-loop and 5hmC changes in significantly differential R-loop regions with overlapping significantly differential 5hmC upon TDP-43 knockdown. Numerals indicate the number of overlapping regions for each group, and numerals in parentheses indicate the number of genes annotated by these regions. (D) Scatterplot of the RNA-seq results for the 226 genes in (C), group I. Significantly downregulated genes (n = 131) are marked in green. (E) Gene Ontology (GO) analysis for significantly downregulated genes with significantly depleted R-loops (n = 379). Comparison of the significant GO terms (FDR < 0.05) for downregulated genes locally co-regulated by R-loop and 5hmC crosstalk (n = 137, y axis) and the remaining genes (n = 242, x axis). (F) Protein-protein interaction network of genes annotated by downregulated R-loops and enriched in the GO term “regulation of growth.” Genes that fall into the list of 137 genes are in green. (G) Visual analysis of R-loops and 5hmC across the NRG1 gene using IGV. (H) RNA-seq results for NRG1 mRNA levels. FPKM, fragments per kilobase of transcript per million mapped reads. (I) DRIP-qPCR and hMe-Seal-qPCR analyses of R-loops and 5hmC in the NRG1 gene body, respectively. Data are plotted as the mean ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t test).
Figure 4.
Figure 4.. TDP-43 knockdown attenuates long-range enhancer/promoter interactions and decreases R-loop and 5hmC levels at a group of enhancers
(A) Screenshot of Micro-C contact map of chromosome 7 (chr7:25,000,000–30,000,000) in shNC and shTDP-43 (n = 2). (B) Volcano plot of the Micro-C results comparing shNC and shTDP-43 at a resolution of 50 kb (diffHic, FDR < 0.05). Differential interactions that can be annotated to enhancer/promoter interactions obtained from EnhancerAtlas 2.0 are in blue and red. (C) Aggregate peak analysis (APA) for the 2,817 significantly downregulated interactions identified in (B). Pixel resolution: 10 kb. (D) Comparison of log2 fold changes in Micro-C read counts within 2,817 significantly lost interactions (50 × 50 kb) with those within the eight surrounding regions (50 × 50 kb). Down, 2,817 significantly downregulated interactions; Surr, the eight surrounding regions. The p value was calculated by Student’s t test. (E) Expression changes of downregulated genes targeted by the significantly downregulated interactions (blue dots in B) and the non-significant (gray dots on the left side of the vertical dashed line and below the horizontal dashed line). The p value was calculated by Student’s t test. (F) Heatmap of the R-loop changes (left) and 5hmC changes (right) in enhancers (n = 1,249) that fall into the significantly downregulated interactions. (G) Expression changes of genes targeted by R-loop- or 5hmC-regulated enhancers. Enhancers in (F) set II were stratified into 10 quantiles according to the fold change in downregulated R-loops or 5hmC, and the expression changes of the genes targeted by either the top 10% or the bottom 10% of the enhancers were analyzed (n = 126, Student’s t test). (H) R-loop changes and 5hmC changes in the top 10% and bottom 10% downregulated genes targeted by the enhancers in (F) set II (n = 126, Student’s t test). (I) Scatterplots of the RNA-seq results for genes targeted by the enhancers in (F) set II. Significantly downregulated genes (n = 94) distally regulated by the crosstalk of R-loop and 5hmC loss at enhancers are highlighted in blue.
Figure 5.
Figure 5.. TDP-43 knockdown regulates the expression of a subset of genes with crucial biological functions, both locally and distally
(A) Protein-protein interaction network of genes regulated by TDP-43-deficiency-mediated R-loop and 5hmC dysregulation locally and distally (n = 217). (B) An example of genes that are regulated by the R-loop-mediated attenuation of enhancer/promoter interactions. IGV shows an ATAC-seq peak at the enhancer region and decreases in R-loop and 5hmC levels. Solid blue lines indicate significantly downregulated interactions, and dashed lines represent the interactions without significant downregulation over the given region. Upregulated genes are marked in red, and downregulated genes are marked in blue. Significantly downregulated genes are marked in bold blue. (C) GOchord plot of the 17 ALS risk genes downregulated by TDP-43-knockdown-mediated R-loop and 5hmC changes, locally and distally, and their participation in multiple biological processes. The colors of the gene names show their regulation by R-loops and 5hmC locally (green), distally (blue), or both (purple). Log2 fold change in the gene expression between shTDP-43 and shNC is shown in blue gradient color.
Figure 6.
Figure 6.. TDP-43 knockdown leads to TE activation, with R-loops and 5hmC contributing to particular TE loci derepression
(A) Volcano plot of the RNA-seq results showing the expression change of TE families upon TDP-43 knockdown (TEtranscripts, n = 3, FDR < 0.05). (B) Circos plot showing the changes in ATAC, R-loop, and 5hmC signals. Track 1: changes in chromatin accessibility at all TE loci with ATAC-seq signals (mean of ATAC-seq read counts across all the samples >5) for the 450 activated TEs. Tracks 2, 3, and 4:the number of up- or downregulated ATAC-seq, R-loop, and 5hmC signals at all TE loci for the 46 activated key TE families with over 50% of loci containing upregulated ATAC/R-loop/5hmC signals (10 Mb resolution). Track 5: 390 loci with upregulation of ATAC-seq/R-loop/5hmC signals for the 46 TE families. Bar plot shows the percentages of TE loci showing upregulation or down-regulation of ATAC-seq/R-loop/5hmC signals for the 46 TE families. (C) Bar plot analysis of the ATAC-seq, R-loop, and 5hmC alterations at the 390 loci (colored bars) for the 46 TE families compared with the other loci (n = 1,662) with upregulation of ATA-seq/R-loop/5hmC signals (gray bars). (D) Boxplot analysis of the expression changes of MER11B and L1MA5 upon TDP-43 knockdown (TEtranscripts, FDR < 0.05). (E) IGV snapshots of R-loop, ATAC-seq, and 5hmC in specific genomic loci of MER11B and L1MA5. (F–H) Volcano plots of the RNA-seq results of shTDP-43 SK-N-DZ cells (PRJEB42763) (F), shTDP-43 iPSC-induced neurons (GSE77702) (G), and iPSC-induced neurons with the ALS-relevant mutation TDP-43Q331K (PRJEB47567) (H). Colored dots indicate the 450 TE families significantly activated in our shTDP-43 cells.
Figure 7.
Figure 7.
TDP-43 chronic deficiency leads to dysregulation of a group of genes and transposable elements via R-loop and 5hmC alteration
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Pinarbasi ES, Cağatay T, Fung HYJ, Li YC, Chook YM, and Thomas PJ (2018). Active nuclear import and passive nuclear export are the primary determinants of TDP-43 localization. Sci. Rep 8, 7083. - PMC - PubMed
    1. Lee EB, Lee VMY, and Trojanowski JQ (2011). Gains or losses: molecular mechanisms of TDP43-mediated neurodegeneration. Nat. Rev. Neurosci 13, 38–50. - PMC - PubMed
    1. Bjork RT, Mortimore NP, Loganathan S, and Zarnescu DC (2022). Dysregulation of Translation in TDP-43 Proteinopathies: Deficits in the RNA Supply Chain and Local Protein Production. Front. Neurosci 16, 840357. - PMC - PubMed
    1. Alami NH, Smith RB, Carrasco MA, Williams LA, Winborn CS, Han SSW, Kiskinis E, Winborn B, Freibaum BD, Kanagaraj A, et al. (2014). Axonal transport of TDP-43 mRNA granules is impaired by ALS-causing mutations. Neuron 81, 536–543. - PMC - PubMed
    1. Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, Bruce J, Schuck T, Grossman M, Clark CM, et al. (2006). Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130–133. - PubMed

Publication types

MeSH terms