Research ArticleCANCER GENETICS

ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis

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Science Advances  20 Jan 2017:
Vol. 3, no. 1, e1601602
DOI: 10.1126/sciadv.1601602
  • Fig. 1 ASXL1 associates with cohesin complex proteins.

    (A) Table of the most relevant proteins identified by LC-MS/MS in the affinity purification of ASXL1-associated proteins using FLAG-ASXL1 overexpressing HEK293T cells. Spectral counts (unique and total) for each interacting protein are shown. (B to E) Reciprocal IP and Western blotting confirmed interaction of ASXL1 with SMC1A, SMC3, and RAD21 in nuclear fraction derived from HEK293T cells transfected with pcDNA3.1+ (Vec) or FLAG-tagged ASXL1 (ASXL1). Nuclear extractions were subjected to IP using indicated antibodies against FLAG (B), SMC1A (C), SMC3 (D), or RAD21 (E). IB, immunoblot. (F) Western blot shows the endogenous interaction between ASXL1 and SMC1A, SMC3, and RAD21 in BM cells of WT mice. IgG, immunoglobulin G. (G) Gel filtration analysis of nuclear extracts from FLAG-ASXL1 overexpressing cells. ASXL1 and the cohesin complex were coeluted from a Superose 6 HR gel filtration column, as analyzed by Western blotting. The numbers over the lanes represent the eluted fraction numbers.

  • Fig. 2 Mapping the region of ASXL1 that mediates its binding to the cohesin complex.

    (A) Schematic diagram of the full-length (FL) ASXL1 and the truncated variants of Asxl1 [amino acids (aa) 1 to 1010, 1 to 420, 1 to 587, and 401 to 587]. Binding affinity was determined by the pull-down efficiency of IP with anti-FLAG and Western blotting with cohesin antibodies. NLS, nuclear localization signal. (B to E) Western blotting analysis of nuclear fractions and anti-FLAG immunoprecipitates from pcDNA3.1+, or each truncated ASXL1 transfected HEK293T cells using antibodies against FLAG, SMC1A, SMC3, or RAD21.

  • Fig. 3 Loss of Asxl1 leads to premature sister chromatid separation in cells.

    (A and B) The myeloid cells with premature sister chromatid separation are frequently seen in PB smears (A) and BM (B) of Asxl1+/− and Asxl1−/− mice with MDS. Red arrows indicate the abnormal nuclear bridging. Scale bars, 5 μm (A) and 10 μm (B). (C and D) Representative cells with premature sister chromatid separation in cultured WT, Asxl1+/−, and Asxl1−/− LK cells. Red arrows indicate the premature sister chromatid separation. The frequency of cells with premature sister chromatid separation is shown in (C). Y axis shows the percentage of cells with premature sister chromatid separation within all binucleated cells. Data are represented as means ± SEM from three independent experiments. ***P < 0.001 and **P < 0.01. Scale bars, 5 μm. (E) The frequency of cells with premature sister chromatid separation in the HeLaGFP-H2B cells with hASXL1 KD and hASXL1 KD plus mAsxl1 rescues. KD of ASXL1 leads to increased frequency of cells with premature sister chromatid separation in HeLaGFP-H2B cells. Reintroducing full-length mAsxl1 rescued the premature sister chromatid separation in HeLa cells with ASXL1 KD. Data are represented as means ± SEM from three independent experiments. ***P < 0.001 and **P < 0.01. (F and G) SMC1A or RAD21 KD leads to premature sister chromatid separation in HeLaGFP-H2B cells. Representative photomicrographs show the cells with premature sister chromatid separation, as indicated by red arrowheads (G). The frequency of cells with premature sister chromatid separation is shown in (F). Y axis shows the percentage of cells with premature sister chromatid separation within all binucleated cells. Data are represented as means ± SEM from three independent experiments. ***P < 0.001 and **P < 0.01. Scale bars, 5 μm. (H) Western blotting shows the expression of full-length ASXL1 and ASXL1 amino acids 401 to 587 in HeLaGFP-H2B cells transfected with vector only, full-length ASXL1, or ASXL1 amino acids 401 to 587. β-Actin serves as loading control. (I and J) ASXL1 amino acids 401 to 587 induce chromatin bridging in HeLaGFP-H2B cells. Quantification of the frequency of cells with premature sister chromatid separation in HeLaGFP-H2B cells transfected with pcDNA3.1+, full-length ASXL1, or ASXL1 amino acids 401 to 587 (J). Data are represented as means ± SEM from three independent experiments. **P < 0.01 for ASXL1 amino acids 401 to 587 fragment versus pcDNA3.1+ or full-length ASXL1. Red arrows indicate the premature sister chromatid separation. Scale bars, 5 μm.

  • Fig. 4 Loss of Asxl1 leads to a decreased cohesin occupancy on the genome but does not affect their DNA recognition sequence.

    (A) Venn diagram showing overlapping peaks between ASXL1, SMC1A, and RAD21 ChIP-seq in WT LK cells. (B) Genomic distribution of ASXL1/SMC1A/RAD21 triple overlapping ChIP peaks in WT LK cells. (C) The overlap analysis shows the peak reads in WT and Asxl1−/− LK cells based on ASXL1/SMC1A/RAD21 overlapping peaks from WT LK cells. Zero base pair (bp) is defined as the peak of ASXL1 binding sites on the genome of WT LK cells. Decreased genomic cohesin complex occupancy is seen in Asxl1−/− LK cells. The overlap peaks of SMC1A and RAD21 represent the cohesin occupancy on the genome. Comparison of the SMC1A/RAD21 overlapping peaks between WT and Asxl1−/− LK cells identified 7833-peak loss and 1175-peak gain in the Asxl1−/− LK cells. TSS, transcription start site. (D) The pie chart represents the percentage of genes with no cohesin occupancy change (remain) or cohesin loss (SMC1A and/or RAD21 peak loss) in Asxl1−/− BM LK cells based on all ASXL1/SMC1A/RAD21 triple overlapping peaks of WT LK cells. (E) DNA recognition sequence of SMC1A and RAD21 in WT or Asxl1−/− LK cells. The SMC1A and RAD21 recognized identical DNA motif as CTCF.

  • Fig. 5 ASXL1 regulates gene expression via the cohesin complex in LK cells.

    (A) The heatmap shows the differentially expressed genes associated with loci of no changes in SMC1A and RAD21 occupancy in Asxl1−/− BM LK cells. (B) The heatmap shows the differentially expressed genes associated with loss of SMC1A and/or RAD21 in Asxl1−/− BM LK cells. (C) The GO analysis of the 237 up-regulated genes (of the ~1600 genes with loss of RAD21 and/or SMC1A occupancy in Asxl1−/− LK cells) in Asxl1−/− LK cells compared to the WT LK cells. (D) GO analysis of the 65 down-regulated genes (of ~1600 genes with loss of RAD21 and/or SMC1A occupancy in Asxl1−/− LK cells) in Asxl1−/− LK cells compared to the WT LK cells. The P values of each GO term are represented by the red dots, and the gene set counts are represented by the bars. (E) Heatmap of differentially expressed genes of myeloid malignancy relevance within Asxl1/SMC1/RAD21 overlapping loci in Asxl1−/− LK cells. (F and G) Genome browser tracks of the Cbfb and Fus locus with overlapping ASXL1, SMC1A, or RAD21 peaks. (H) Relative RNA level of Cbfb, Fus, and Stat3 in LK cells as determined by qPCR. Data are represented as means ± SEM from three independent experiments. ***P < 0.001 and **P < 0.01.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/1/e1601602/DC1

    fig. S1. ASXL1 forms a complex with the cohesin complex.

    fig. S2. Reintroducing mAsxl1 rescued the premature sister chromatid separation in HeLa cells with ASXL1 KD.

    fig. S3. Enrichment map was used for visualizing the network of selected GO terms enriched with up-regulated and down-regulated genes in Asxl1−/− LK cells.

    table S1. List of ASXL1 interaction proteins identified by MS in HEK293T cells transfected with FLAG-ASXL1.

    table S2. qPCR primer sequences.

    table S3. Statistical evidence for binding between SMC1A, RAD21, and ASXL1.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. ASXL1 forms a complex with the cohesin complex.
    • fig. S2. Reintroducing mAsxl1 rescued the premature sister chromatid separation in HeLa cells with ASXL1 KD.
    • fig. S3. Enrichment map was used for visualizing the network of selected GO terms enriched with up-regulated and down-regulated genes in Asxl1−/− LK cells.
    • Legend for table S1
    • table S2. qPCR primer sequences.
    • table S3. Statistical evidence for binding between SMC1A, RAD21, and ASXL1.

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    Other Supplementary Material for this manuscript includes the following:

    • table S1 (Microsoft Excel format). List of ASXL1 interaction proteins identified by MS in HEK293T cells transfected with FLAG-ASXL1.

    Files in this Data Supplement:

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