Region-specific alteration of histone modification by LSD1 inhibitor conjugated with pyrrole-imidazole polyamide

Abstract

Epigenome regulates gene expression to determine cell fate, and accumulation of epigenomic aberrations leads to diseases, including cancer. NCD38 inhibits lysine-specific demethylase-1 (LSD1), a histone demethylase targeting H3K4me1 and H3K4me2, but not H3K4me3. In this study, we conjugated NCD38 with a potent small molecule called pyrrole (Py) imidazole (Im) polyamide, to analyze whether targets of the inhibitor could be regulated in a sequence-specific manner. We synthesized two conjugates using β-Ala (β) as a linker, i.e., NCD38-β-β-Py-Py-Py-Py (NCD38-β2P4) recognizing WWWWWW sequence, and NCD38-β-β-Py-Im-Py-Py (NCD38-β2PIPP) recognizing WWCGWW sequence. When RKO cells were treated with NCD38, H3K4me2 levels increased in 103 regions with significant activation of nearby genes (P = 0.03), whereas H3K4me3 levels were not obviously increased. H3K27ac levels were also increased in 458 regions with significant activation of nearby genes (P = 3 × 10−10), and these activated regions frequently included GC-rich sequences, but less frequently included AT-rich sequences (P < 1 × 10−15) or WWCGWW sequences (P = 2 × 10−13). When treated with NCD38-β2P4, 234 regions showed increased H3K27ac levels with significant activation of nearby genes (P = 2 × 10−11), including significantly fewer GC-rich sequences (P < 1 × 10−15) and significantly more AT-rich sequences (P < 1 × 10−15) compared with NCD38 treatment. When treated with NCD38-β2PIPP, 82 regions showed increased H3K27ac levels, including significantly fewer GC-rich sequences (P = 1 × 10−11) and fewer AT-rich sequences (P = 0.005), but significantly more WWCGWW sequences (P = 0.0001) compared with NCD38 treatment. These indicated that target regions of epigenomic inhibitors could be modified in a sequence-specific manner and that conjugation of Py-Im polyamides may be useful for this purpose.

Py 3 -FITC: a new fluorescent probe for live cell imaging of collagen-rich tissues and ionocytes

“…In zebrafish embryos, several types of ionocytes, such as H + -ATPase-rich (HR) cells, Na + -K + -ATPase-rich (NaR) cells, Na + -Cl − cotransporter (NCC) cells, solute carrier 26 (SLC26) cells and K + -secreting (KS) cells, are in the skin and gill [15]. HR cells secrete acid, take up Na + and excrete NH 4 + and are similar to mammalian renal proximal tubular cells [15]. Therefore, an easy imaging tool for HR cells may make these cells useful as model systems of mammalian renal proximal tubular cells.…”

“…Long-length poly-Py-based polyamides are not always effective. Inversely, short polyamides could bind multiple genomic regions [4]. The addition or deletion of a single charge might change the nuclear uptake effect [5].…”