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 mechanistic role as genomic insulators, shielding the Tyr locus from the expression patterns of adjacent genes.
Highlights
Control of gene expression is dictated by cell-type specific regulatory sequences that physically organize the structure of chromatin, including promoters, enhancers and insulators
The mouse Tyr locus spans over 180 kb on chromosome 7 and its expression patter is restricted to melanocytes and in cells of the retinal pigmented epithelium[24]
The anchor primer was positioned at the CNS-242, a sequence observed to be dispensable for Tyr expression in transgenic mice generated with different yeast artificial chromosomes (YACs) Tyr transgenes[31]
Summary
Control of gene expression is dictated by cell-type specific regulatory sequences that physically organize the structure of chromatin, including promoters, enhancers and insulators. Mutations at Tyr affect coat and eye pigmentation, with similar alterations in the visual pathway[13], but without gross detrimental effects on overall physiology and viability For this reason, Tyr has been targeted in m ouse[14] and other o rganisms[15–17] to benchmark transgenesis and genome engineering strategies, from oocyte microinjection of DNA18,19 to the most recent CRISPR nuclease[20] and base editor approaches[21,22]. A regulatory element 5′ upstream in the murine Tyr locus was first identified as a DNAse I hypersensitivity s ite[28] The inclusion of this element in a tyrosinase minigene resulted in copy number-dependant gene expression in transgenic mice[29], suggesting features of a chromatin b oundary[30] or a locus control region (LCR)[31,32]. Using in vivo mouse studies, we resolve the chromatin structure of the Tyr locus and integrate it with functional data obtained by targeting additional putative cis-regulatory sequences at the 3′ downstream of the Tyr gene
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