MKRN3 Interacts With Several Proteins Implicated in Puberty Timing but Does Not Influence GNRH1 Expression

Abstract

Paternally-inherited loss-of-function mutations in makorin ring finger protein 3 gene (MKRN3) underlie central precocious puberty. To investigate the puberty-related mechanism(s) of MKRN3 in humans, we generated two distinct bi-allelic MKRN3 knock-out human pluripotent stem cell lines, Del 1 and Del 2, and differentiated them into GNRH1-expressing neurons. Both Del 1 and Del 2 clones could be differentiated into neuronal progenitors and GNRH1-expressing neurons, however, the relative expression of GNRH1 did not differ from wild type cells (P = NS). Subsequently, we investigated stable and dynamic protein-protein interaction (PPI) partners of MKRN3 by stably expressing it in HEK cells followed by mass spectrometry analyses. We found 81 high-confidence novel protein interaction partners, which are implicated in cellular processes such as insulin signaling, RNA metabolism and cell-cell adhesion. Of the identified interactors, 20 have been previously implicated in puberty timing. In conclusion, our stem cell model for generation of GNRH1-expressing neurons did not offer mechanistic insight for the role of MKRN3 in puberty initiation. The PPI data, however, indicate that MKRN3 may regulate puberty by interacting with other puberty-related proteins. Further studies are required to elucidate the possible mechanisms and outcomes of these interactions.

Keywords: CRISPR/Cas9; GNRH1; MKRN3; protein interaction; puberty timing.

Figure 1

CRISPR/Cas9-based generation of two bi-allelic MKRN3 KO cell lines (Del 1 and Del 2) in human pluripotent stem cell line H9 and verification of CRISPR/Cas9 induced deletions at genomic DNA level. (A) Left panel, Del 1 has a deletion of 174 bps, translating to a deletion of 51 amino acids (11% of protein) including the translation start site. Right panel, Del 2 has a deletion of 901 bps, translating to 300 amino acid deletion (60% of protein). (B) The presence or absence of CRISPR/Cas9-induced deletions in MKRN3 was verified by PCR amplification of the region of interest from genomic DNA of WT human pluripotent H9 cells, and in CRISPR/Cas9 edited cells. Four examples of edited cell lines are shown (Del 1–Del 4). The same pair of guide RNAs, designed to delete ~900 bps, was used for editing Del 2 and Del 3 cell lines, whereas two different gRNA pairs were used to edit Del 1 and Del 4 cell lines. The PCR products were visualized on a 1.5% agarose gel.

Figure 2

(A) Schematic of MKRN3 cDNA and the two pairs of primers (green and blue arrows) employed in detection of WT, Del 1, (left panel) and Del 2, (right panel). cell lines. In WT cells, the expected product sizes are 305 and 1,235 bp, in Del 1 cells 131 bp and in Del 2 cells 334 bp. (B) MKRN3 amplification from the cDNA of the two bi-allelic MKRN3 deletion cell lines Del 1, (left panel) and Del 2, (right panel). White arrow indicates the expected wild type (WT) product sizes (305 and 1,235 bp, respectively) and the red arrows indicate the expected sizes (131 and 334 bp, respectively) for the PCR products from Del 1 and Del 2 cell lines. The cDNA used is from day 25 of the protocol generating GNRH1-expressing neurons. The PCR products were visualized on a 1.5% agarose gel.

Figure 3

(A) Schematic for the generation of GNRH1-expressing neurons from hPSCs (17). In brief, dual SMAD inhibition by Dorsomorphin and SB431542 was applied for the first 10 days, followed by 10 days of FGF8 treatment and final stage of neuronal maturation for 5–7 days by combining FGF8 with Notch inhibition by DAPT. This differentiation methodology has been adapted from a previously published protocol and the figure has been modified from the original publication by Lund et al. (17). (B) Immunocytochemistry in WT and Del 2 derived day 25 GnRH-positive neurons (green) nuclear stained with DAPI (blue), scale bars represent 100 μM. (C) The relative expression of GNRH1 in WT H9 cells (n = 9) and in two bi-allelic MKRN3 KO cell lines (Del 1 and Del 2) following three independent repeats (n = 6) of differentiation into GNRH1-expressing neurons (17). Expression levels are relative to day 0 hPSCs.

Figure 4

Protein-protein interaction map of MKRN3. The network represents PPIs of MKRN3 detected by different purification methods. Blue edges represent experimentally validated interaction by AP-MS; Red lines represent interactions detected by BioID purification; Overlap of two purification methods is shown with gray color, while a dashed line describes prey-prey interactions. The colored nodes represent the different clusters of prey proteins.