Research ArticleMOLECULAR BIOLOGY

ALBA protein complex reads genic R-loops to maintain genome stability in Arabidopsis

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Science Advances  15 May 2019:
Vol. 5, no. 5, eaav9040
DOI: 10.1126/sciadv.aav9040
  • Fig. 1 AtALBA1 and AtALBA2 bind R-loops in vitro.

    (A) EMSA gel showing AtALBA1 binding to ssRNA and DNA-RNA hybrids. Different 5′-biotin–labeled substrates (5 nM) were incubated with increasing concentrations (25, 50, and 75 nM) of AtALBA1 wild-type protein (lanes 2 to 4) and 75 nM AtALBA1 (K30E) mutant protein (lane 5). (B) EMSA gel showing AtALBA2 binding to ssDNA and dsDNA. Different 5′-biotin–labeled substrates (5 nM) were incubated with increasing concentrations (25, 50, and 75 nM) of AtALBA2 wild-type protein (lanes 2 to 4) and 75 nM AtALBA2 (K30E) mutant protein (lane 5). (C) EMSA gel showing AtALBA1 binding to artificial R-loops. Artificial R-loop substrate (5 nM) with 5′-biotin–labeled DNA (1) or RNA (2) was incubated with 75 nM AtALBA1 wild-type protein. R-loop substrates were incubated with RNase H1 for 0 min and 10 min. (D) EMSA gel showing AtALBA2 binding to artificial R-loops. Artificial R-loop substrate (5 nM) with 5′-biotin–labeled DNA (1) or RNA (2) was incubated with 75 nM AtALBA2 wild-type protein. R-loop substrates were incubated with RNase H for 0 and 10 min. For EMSAs, at least three biological replicates were performed, and representative results are shown.

  • Fig. 2 AtALBA1 preferentially binds gene body regions with active epigenetic marks in vivo.

    (A) Total number and genomic distribution of AtALBA1 peaks identified by ChIP-seq. (B) Metagene plots of AtALBA1 ChIP-seq reads. TSS, transcription start site; TTS, transcription terminal site; −2 K and +2 K represent 2 kb upstream of the TSS and 2 kb downstream of the TTS, respectively. The y axis indicates AtALBA1 ChIP-seq read density. (C) Length distribution of AtALBA1-bound genes. The y axis indicates the number of genes. The x axis indicates the length of genes. (D) Metagene plots of histone modification levels on AtALBA1-bound genes. The y axis represents histone modification ChIP-seq read density. (E) The relationship between AtALBA1 and AtALBA2 binding and repressive histone modifications was determined by immunostaining. AtALBA1-Flag and AtALBA2-Flag in transgenic plants were stained with anti-Flag (red). H3K9me1 was stained with anti-H3K9me1 (green). DNA was stained with DAPI (blue). The frequency of nuclei displaying each interphase pattern is shown on the right. Scale bar, 2.5 μm.

  • Fig. 3 AtALBA1 and AtALBA2 binding correlates with the presence of R-loops.

    (A) Metagene plots of R-loop levels across AtALBA1-bound genes. The y axis indicates ssDRIP-seq read density. (B) Percentages of AtALBA1-bound genes overlapping with sense, antisense, and overlap (sense and antisense) R-loops. Enrichment ratio of AtALBA1-bound genes harboring overlap R-loops to all genes harboring overlap R-loops in the Arabidopsis genome was indicated. P value was calculated with R from Fisher’s exact test. (C) Association of AtALBA1 and AtALBA2 with R-loops determined by ChIP-qPCR. Transgenic AtALBA1-Flag/alba1-1 and AtALBA2-Flag/alba2-1 plants were used. Expression of AtALBA1-Flag and AtALBA2-Flag was under the control of their respective native promoters. ChIP experiments were performed with anti-Flag antibody. The RNase H treatment was performed before cross-linking. Genes overlapping with sense, antisense, and overlap R-loops were represented by red, blue, and yellow colors, respectively. An intergenic region without R-loop formation is chosen as a negative control. Two biological replicates yielded very similar results. SEs were calculated from three technical replicates; *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t test).

  • Fig. 4 Depletion of AtALBA1 or AtALBA2 results in plant hypersensitivity to MMS.

    (A) Representative microscopic images showing γH2AX foci formation (green) in Col-0, alba1-1, alba1-2, alba2-1, alba1-1alba2-1, and alba1-2alba2-1 plants treated with 50 ppm of MMS. γH2AX foci were detected by immunostaining using an anti-γH2AX antibody. Nuclei were stained with DAPI (blue). Scale bars, 5 μm. (B) Box plots showing the signal intensity of γH2AX foci per nucleus for Col-0 plants and the indicated mutants. The γH2AX signal intensity was analyzed by ImageJ software. Dark horizontal line, median; edges of boxes, 25th (bottom) and 75th (top) percentiles; whiskers, minimum and maximum gray values. The multiple comparison was calculated with Kruskal-Wallis. The α parameter by default is 0.05. Post hoc test used the criterium Fisher’s least significant difference. The adjustment methods include the Bonferroni correction and others. (C) Fresh weights of 14-day-old Col-0 seedlings and the indicated mutant seedlings grown on 1/2 MS medium supplemented with 0 or 20 ppm of MMS. The fresh weights of 120 seedlings were statistically analyzed. SEs were calculated from three biological replicates; *P < 0.05, **P < 0.01 (two-tailed Student’s t test). (D) Metaplot of γH2AX accumulation in AtALBA1-bound regions (solid lines) versus randomly selected regions (dash lines) in Col-0 and alba1-1alba2-1 after MMS treatment. (E) A working model for the role of AtALBA1 and AtALBA2 in R-loop biology. AtALBA1 and AtALBA2 form a heterodimer or heteropolymer and bind R-loops at genic regions with active histone marks. By occupying R-loops, AtALBA1 and AtALBA2 protect R-loops from DNA damage and help maintain genome stability.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaav9040/DC1

    Fig. S1. Domain structure of ALBA proteins in Arabidopsis.

    Fig. S2. Purification of AtALBA1 and AtALBA2 wild-type and mutant proteins and diagram of probes used in EMSAs.

    Fig. S3. Characterization of the nucleic acid binding properties of AtALBA1 and AtALBA2.

    Fig. S4. Subcellular localization and interaction of AtALBA1 and AtALBA2.

    Fig. S5. Characterization of AtALBA1-bound loci.

    Fig. S6. Characterization of the T-DNA insertion mutants for AtALBA1 and AtALBA2.

    Fig. S7. Detection of R-loop levels in Col-0 and alba1-1alba2-1 by immunostaining and ssDRIP-seq.

    Fig. S8. Molecular phenotypes of Col-0 and alba1-1alba2-1 without and with MMS treatment.

    Table S1. Primers and substrates used in this study.

    Table S2. List of AtALBA1-bound loci.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Domain structure of ALBA proteins in Arabidopsis.
    • Fig. S2. Purification of AtALBA1 and AtALBA2 wild-type and mutant proteins and diagram of probes used in EMSAs.
    • Fig. S3. Characterization of the nucleic acid binding properties of AtALBA1 and AtALBA2.
    • Fig. S4. Subcellular localization and interaction of AtALBA1 and AtALBA2.
    • Fig. S5. Characterization of AtALBA1-bound loci.
    • Fig. S6. Characterization of the T-DNA insertion mutants for AtALBA1 and AtALBA2.
    • Fig. S7. Detection of R-loop levels in Col-0 and alba1-1alba2-1 by immunostaining and ssDRIP-seq.
    • Fig. S8. Molecular phenotypes of Col-0 and alba1-1alba2-1 without and with MMS treatment.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Primers and substrates used in this study.
    • Table S2 (Microsoft Excel format). List of AtALBA1-bound loci.

    Files in this Data Supplement:

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