SASH1 S519N Variant Links Skin Hyperpigmentation and Premature Hair Graying to Dysfunction of Melanocyte Lineage

Abstract

A better understanding of human melanocyte (MC) and MC stem cell biology is essential for treating MC-related diseases. This study employed an inherited pigmentation disorder carrying the SASH1^S519N variant in a Hispanic family to investigate SASH1 function in the MC lineage and the underlying mechanism for this disorder. We used a multidisciplinary approach, including clinical examinations, human cell assays, yeast 2-hybrid screening, and biochemical techniques. Results linked early hair graying to the SASH1^S519N variant, a previously unrecognized clinical phenotype in hyperpigmentation disorders. In vitro, we identified SASH1 as a regulator in MC stem cell maintenance and discovered that TNKS2 is crucial for SASH1's role. In addition, the S519N variant is located in one of multiple tankyrase-binding motifs and alters the binding kinetics and affinity of the interaction. In summary, this disorder links both gain and loss of pigmentation in the same individual, hinting to accelerated aging in human MC stem cells. The findings offer insights into the roles of SASH1 and TNKS2 in MC stem cell maintenance and the molecular mechanisms of pigmentation disorders. We propose that a comprehensive clinical evaluation of patients with MC-related disorders should include an assessment and history of hair pigmentation loss.

Keywords: Aging; Human melanocyte stem cell maintenance; Lentigines; Tankyrase 2; Tankyrase-binding motif.

Figure 1.. Increased numbers of non-proliferating MCs in the skin of SASH1^S519N individuals.

(a) Immunofluorescent (IF) staining of skin biopsies from unaffected control individuals (WT) or individuals carrying S519N. Scale bar = 100 um. (b) shows the number of MCs (TYR+ cells; n = 140 for controls and 389 for S519N), and (c) shows the percentage of proliferating MCs (TYR+KI67+/TYR+ × 100; n = 21 for controls and 13.5 for S519N), with 6 individuals in the WT group (C1–6) and 3 lesional (L1–3) and 3 non-lesional (NL1–3) biopsies in the S519N group. (d) shows SASH1 (WT or S519N) does not activate RAS/MAPK signaling in human MC cultures. An immortalized human melanocyte cell line, PIG1 was transfected with control empty vector (EV) or SASH1 (WT or S519N) +/− activated NRAS^Q61K prior to immunoblot.

Figure 2.. SASH1^WT but not SASH1^S519N promoted sphere forming capacity in cells and SASH1^S519N accelerated hair graying in humans.

Human primary epidermal melanocyte cells (HEMn-MP) were transfected with control vector (EV), WT SASH1 or S519N. Cells were then subjected to the sphere forming assay (a and b), Aldefluor assay (c), or immunoblot (d). (e) Early onset of hair-graying in the S519N individuals. Percent members from WT (0/8) or S519N family members (8/11) with onset of hair-graying at ≤ or > 20 years old. (f) Examples of hair in individuals carrying WT or S519N of SASH1. Subjects consented to the publication of these images.

Figure 3.. SASH1 requires TNKS2 for its function.

(a) Y2H screens identified top binders to SASH1 using full length human SASH1 (WT or S519N) as bait and a human MC generated cDNA library as the prey. (b) S519N is in a predicted TBM. (c) SASH1 has nine predicted TBMs. (d) Human primary MCs were transfected with expression vectors of SASH1 (control vector EV, WT, or S519N), with siRNAs against TNKS2 or a si-Scramble control, and 60–70% knockdown was achieved (Supplementary Figure S2). Cells were then subjected to sphere forming assays. (e) Locations of the TBMs and the variants associated with pigmentation disorders in SASH1. (f) The SPIDER region is conserved within vertebrates, contains four TBMs (highlighted sequences), and most hyperpigmentation variants are in or close to TBM6.

Figure 4.. S519N is within one of four validated TBMs in SPIDER.

(a) Co-IP with HA-tagged magnetic beads showed pull-down of TNKS2 by SASH1 from co-transfected HA-tagged SASH1 (EV, WT, S519N) and TNKS2 (EV or TNKS2) in 293FT cells. (b) ¹⁵N-¹H HSQC spectrum of WT SASH1-SPIDER (396–564) alone (gray) and with 12x concentration TNKS2-ARC4 (red). Three backbone chemical shifts from TBM6 are expanded to represent perturbations (CSPs) (L515) or peak broadening (G517, S519). (c) and (d) CSPs for WT and S519N SPIDER at 4x concentration ARC4. Yellow bars represent predicted TBMs, and dotted lines represent the averages plus one standard deviation, 0.031 and 0.029 ppm. (e) Overlay of normalized peak intensity for both WT and S519N SPIDER at 12x concentration.

Figure 5.. The S519N variant alters binding kinetics and decreases binding affinity to TNKS2.

(a) Left, examples of CSPs, up to 1:12, show changes between WT and S519N. Right, overlaid K_D traces with regression-fitted values of both WT (black) and S519N (red). (b) Select CSPs demonstrate an increased peak broadening rate in WT compared to S519N. WT F411, G517, and F555 all immediately broaden and disappear by the 1:1 titration point, but broaden at a higher titration point in S519N. Stars in F555 represent peak splitting. The last visible titration ratio is inset in the bottom corner of the spectra in red. (c) MST dose-response curves for WT (blue) and S519N (red) mixed with fluorescently labeled ARC4. The KD value for the SPIDER WT is 1.7 ± 0.4 μM. K_D value for SPIDER S519N could not be estimated due to weak binding.

Figure 6.. RNAseq indicates a role of SASH1 in mitosis.

Human primary MCs were transfected with si-Control or si-SASH1, cultured in sphere conditions before being subjected to RNAseq (a and b). (a) Volcano plot shows the differential expressed genes between si-SASH1 versus si-Control. (b) GSEA enrichment plots for the top differentiated expressed Hallmark pathways: G2M Checkpoint (p = 0.0004, FDR = 0.044), E2F targets (p = 0.002, FDR = 0.033), Mitotic spindle (p = 0.001, FDR = 0.056). (c) We propose: SASH1 promotes the proper maintenance of the McSC pool and keeps a proper balance between McSC and MC. The S519N variant disrupts McSC maintenance and shifts the balance to more differentiated MCs, resulting in accelerated age-dependent pigmentation phenotypes, including hair graying and lentigines.