Targeted Disruption of the Cohesin Subunit STAG2 Leads to Loss of Insulation and Inappropriate Gene Activation in Response to Differentiation Signals

Cohesin is a multiprotein complex that mediates sister chromatid cohesion during S phase of the cell cycle, but it also plays crucial roles in genome organization and transcription in interphase. Cohesin's role in regulation of the genome helps determine lineage identity and differentiation of hematopoietic cells (Viny et al., JEM, 2015; Mullenders et al., JEM, 2015; Mazumdar et al., Cell stem cell, 2015; Galeev et al., Cell reports, 2016). Mitotic cohesin is composed of RAD21, SMC1A, SMC3 and STAG1/STAG2 subunits. Mutations in the subunits of cohesin complex are present in significant proportion of myeloid malignancies (Thota et al., Blood 2014). In particular, the frequency of cohesin subunit mutation is as high as ~50% in Down-syndrome associated megakaryoblastic leukemia subtype (Yoshida et al., Nature Genetics, 2013).

To further understand cohesin's role in gene regulation in a leukemia context, we used CRISPR-CAS9 to engineer a leukemia associated STAG2 mutation, R614*, into K562 leukemia cells to generate isogenic model lines. Homozygous mutant K562 cells showed complete loss of STAG2 expression. We also obtained STAG2 R614* heterozygous cell clones that showed partial loss of STAG2 expression (STAG2 is on the X chromosome, and the wild type allele is mosiacally silenced in heterozygotes).

Phenotype analyses of STAG2 mutant K562 cells showed that homozygous mutants had altered morphology and had acquired adherent properties. Homozygous mutant cells activated expression of the stem cell marker, KIT, and showed a reduction in the neutrophil differentiation marker, CD15.

We used ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) to examine changes in chromatin accessibility, and RNA sequencing to determine changes in gene expression in STAG2 mutants. More than 5,000 genes were dysregulated in STAG2 homozygous mutants, accompanied by profound changes in chromatin accessibility with gain and loss of accessibility represented equally. Loss of accessibility was observed at insulators that recruit CCCTC-binding factor (CTCF), indicating potential loss of insulation at chromatin boundaries. Conversely, gain of chromatin accessibility was observed at regions that are normally repressed in K562, and this was accompanied by activation of transcription factors such as ERG and FLI1. Gain of accessibility was also observed at myeloid-associated super enhancers of the RUNX1 gene.

We hypothesized that loss of STAG2 leads to an inability to constrain transcriptional programs upon differentiation of leukemia cells. To test this hypothesis, we stimulated the STAG2 mutant and parental K562 cells with phorbol myristate acetate (PMA) to induce megakaryocyte differentiation. PMA-stimulated STAG2 homozygous mutant cells showed a striking precocious spike in RUNX1 expression that was associated with enhanced transcription from its proximal P2 promoter. A similar precocious spike was observed in transcription of ERG. Interestingly, the spike in transcription of these genes was not observed at 48 hours, indicating that the ectopic transcription of RUNX1 and ERG is confined to early response to stimulation. The BET inhibitor JQ1, was previously shown to block enhancer activity (Bhagwat et al., Cell reports, 2016). Treatment of STAG2 mutant cells with JQ1 dampened ectopic RUNX1 P2 expression and led to a complete loss of RUNX1 P1 and ERG activation during PMA stimulation. These results suggest that ectopic RUNX1 expression in response to STAG2 deficiency is enhancer-driven. Furthermore, JQ1 treatment caused a 2-fold decrease in KIT levels in STAG2-deficient cells, indicating a reversal of immature properties in these cells.

In summary, our results suggest that STAG2 mutation results in loss of chromatin insulation, leading to inappropriate activation of hematopoietic enhancers. Loss of constraints on enhancer-driven transcription could in turn underlie the aberrant gene expression in STAG2-deficient cells exposed to differentiation cues.