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. 2020 Oct 13;9:e57117. doi: 10.7554/eLife.57117

Figure 1. HELLS interacts with PRDM9.

(A) Domains of PRDM9 and HELLS PRDM9 includes a Krüppel-associated box domain (KRAB), a synovial sarcoma-X break point-repression domain (SSXRD), a Su(var)3–9, Polycomb-group protein enhancer of zeste and trithorax-group protein TRX domain (PR/SET) that is preceded and followed by zinc-finger domains (ZK and ZF, respectively), and a C2H2-type zinc-finger array (C2H2 ZF array). HELLS contains a coiled-coil domain, a helicase ATPase domain, and a helicase C-terminal domain. (B) Interaction between PRDM9 and HELLS by yeast two-hybrid assays. Full-length and four fragments of mouse HELLS were used to test for interaction with mouse PRDM9 (full length). HELLS domains were fused to the Gal4 activation domain (GAD), and PRDM9 was fused to the Gal4 DNA-binding domain (GBD). A positive interaction was detected for full-length HELLS and fragment 1–569. Growth was tested on medium without leucine and tryptophan (LW), without leucine, tryptophan and histidine (LWH), and without leucine, tryptophan and histidine with 5 mM amino-triazole (LWH + 5 mM AT). A diploid strain that expresses pGAD-REC114 and pGBD-MEI4 (Kumar et al., 2010) was used as positive control. The HELLS region of the cDNAs isolated by yeast two-hybrid screening is shown. Controls are shown in Figure 1—figure supplement 1.

Figure 1.

Figure 1—figure supplement 1. Controls for yeast two-hybrid assays.

Figure 1—figure supplement 1.

(A) Negative controls for the different mouse HELLS domains analyzed by yeast two-hybrid assays. Growth of diploid strains that express the indicated HELLS construct or the empty GBT9 vector were tested on medium without leucine and tryptophan (LW) and on medium without leucine, tryptophan and histidine with 5 mM amino-triazole (LWH + 5 mM AT). Positive control was a diploid strain that expresses pGAD-REC114 and pGBD-MEI4. (B) Western blot analysis of the GAD-HELLS and GBD-PRDM9 fusion proteins expressed in the various diploid strains used in the yeast two-hybrid assays. Blots were probed with anti-GAD, anti-GBD, or anti-tubulin (internal control) antibodies, as indicated.