pH controlled histone acetylation amplifies melanocyte differentiation program downstream of MITF

Abstract

22 differentiation, epigenetics and zebrafish 23 24 Running title: pH regulates melanocyte maturation 25 pH regulates melanocyte maturation 2 Abstract 26 Tanning response and melanocyte differentiation are mediated by the central transcription factor MITF. 27Enigmatically, these involve rapid and selective induction of melanocyte maturation genes, while 28 concomitantly maintaining the expression of other effectors. In this study using cell-based and zebrafish 29 model systems, we elucidate a pH mediated feed-forward mechanism of epigenetic regulation that enables 30 selective amplification of melanocyte maturation program. We demonstrate that MITF activation directly 31 elevates the expression of Carbonic Anhydrase 14 (Ca14) enzyme. Nuclear localized Ca14 increases the 32 intracellular pH, resulting in the activation of histone acetyl transferase activity of p300/CBP. In turn 33 enhanced H3K27 histone acetylation marks of select differentiation genes facilitates their amplified 34 expression by MITF. CRISPR-mediated targeted missense mutation of CA14 in zebrafish results in 35 immature acidic melanocytes with decreased pigmentation, establishing the centrality of this mechanism 36 in rapidly activating melanocyte differentiation. Thereby we reveal a novel epigenetic control through pH 37 modulation that reinforces a deterministic cell fate by altering chromatin dynamics. 38 39 pH regulates melanocyte maturation 102 pH regulates melanocyte maturation 6 Results 103 Alkaline intracellular pH induces pigmentation by enhancing the melanogenesis gene expression 104During the culture of B16 cells in DMEM medium CO 2 -HCO 3 buffering system maintains media pH. 105Hence we resorted to establish the pH-pigmentation link by modulating the prevailing CO 2 levels. We set 106 up progressive pigmentation using the B16 cells as described earlier (Natarajan et al, 2014). In this set up 107 the cells progressively activate the melanogenesis gene expression program and increase pigmentation 108 over a course of 8 to 12 days. To alter the pH, 10% CO 2 levels were tested. We measured the extracellular 109 pH (pH e ) using the standard pH electrode under controlled conditions of temperature and CO 2 saturation. 110While the cells grown in 5% CO 2 showed an increase in pH e from around 7.4 to 7.8 on day 4 after 111 induction of the pigmentation program, the 10% CO 2 sustained a constant pH of around 7.4 112( Supplementary Fig 1A). This trend was reflected in the intracellular pH (pH i ), which is close to 7.9 on 113 day 4 under the routine 5% CO 2 condition. However, under the 10% CO 2 the pH remained close to 7.0, 114 similar to the day 0 where the cells are depigmented (Fig 1A). When the cells were assessed for the 115 cumulative accumulation of melanin on day 8, under the 10% CO 2 condition we observed depigmented 116 cells (Fig 1B). Melanin content assay performed using synthetic melanin standard confirmed that the 117 level of melanin is significantly low (Supplementary Fig S1B). Further electron microscopic evaluation 118 of day...