Boundary sequences flanking the mouse tyrosinase locus ensure faithful pattern of gene expression

Abstract

Control of gene expression is dictated by cell-type specific regulatory sequences that physically organize the structure of chromatin, including promoters, enhancers and insulators. While promoters and enhancers convey cell-type specific activating signals, insulators prevent the cross-talk of regulatory elements within adjacent loci and safeguard the specificity of action of promoters and enhancers towards their targets in a tissue specific manner. Using the mouse tyrosinase (Tyr) locus as an experimental model, a gene whose mutations are associated with albinism, we described the chromatin structure in cells at two distinct transcriptional states. Guided by chromatin structure, through the use of Chromosome Conformation Capture (3C), we identified sequences at the 5' and 3' boundaries of this mammalian gene that function as enhancers and insulators.By CRISPR/Cas9-mediated chromosomal deletion, we dissected the functions of these two regulatory elements in vivo in the mouse, at the endogenous chromosomal context, and proved their role as genomic insulators, shielding the Tyr locus from the expression patterns of adjacent genes.

The structure and function of the mouse tyrosinase locus

“…This observation suggested that the variegated phenotype previously reported in the YAC YRT4, YRT5, and YRT2ΔLCR transgenic mice could be explained by the randomly integrated transgenes, and consequent chromosomal position effects. This experiment also highlighted the relevance of assessing the role of non‐coding DNA regulatory elements in their natural context, an approach that became possible with the arrival of CRISPR‐Cas9 genome‐editing methods (Seruggia et al., 2020; Seruggia et al., 2015; Seruggia & Montoliu, 2016).…”

“…The alignment of the deleted DNA fragments in several independent lines with the same hypopigmented phenotype pointed again to the core element of this 5’ region, including the previously identified hs1/hs2 (box A and B) binding sites for nuclear factors. A detailed histological analysis of the retina from the genome‐edited mice indicated that only the neural crest‐derived melanocytes in the choroid lost Tyr expression and hence showed no pigmentation (Seruggia et al., 2020). By contrast, the RPE layer of cells was normally and uniformly pigmented, thus confirming the notion that the 5’ regulatory element mainly controls Tyr expression in the melanocyte lineage, not in the optic cup‐derived RPE cells.…”

“…Evidence for the existence of a similar genomic boundary at the 3’ of the mouse Tyr locus has emerged from more recent biochemical studies. In particular, the application of Chromosome Conformation Capture (3C) techniques (Hagège et al, 2007), using the Tyr promoter sequences as an anchorage, revealed a strong interaction of the Tyr promoter with DNA sequences mapping at the 3’ untranslated region of the Tyr locus, in both melanoma cells (expressing Tyr ) and fibroblasts (not expressing Tyr ) (Seruggia et al., 2020). Closer observation of these DNA sequences revealed the presence of binding sites for the nuclear factor CTCF, known to transduce insulator activities (Bell et al., 1999).…”

“…To assess the functional relevance of this 3’ Tyr boundary, we generated genome‐edited mice using CRISPR‐Cas9 following an analogous experiment as previously done with the 5’ Tyr boundary (Seruggia et al., 2015). The resulting genome‐edited mice lacking the 3’ Tyr boundary (termed TYRINS3) were indistinguishable from wild‐type pigmented mice, both for coat color and eye pigmentation (Seruggia et al., 2020) (Figure 1). This unexpected phenotype was further investigated by measuring not only Tyr but also Nox4 and Grm5 expression levels in various organs of genome‐edited TYRINS5 and TYRINS3 mice carrying homozygous deletions at the 5’ and 3’ Tyr boundaries, respectively.…”

HPS11 and OCA8: Two new types of albinism associated with mutations in BLOC1S5 and DCT genes

“…Describing new genes associated with albinism will also help to reduce the significant number (~30%) of clinically diagnosed people with albinism, where we fail to detect the responsible molecular lesion (Lasseaux et al., 2018; Montoliu et al., 2014). An additional source for detecting new mutations is the non‐coding DNA regulatory elements, which might also contribute to the missing mutations in these undiagnosed patients (Seruggia et al., 2020).…”