The X chromosome-encoded histone demethylase UTX (also known as KDM6A) mediates removal of repressive trimethylation of histone H3 lysine 27 (H3K27me3) to establish transcriptionally permissive chromatin. Loss of UTX in female mice is embryonic lethal. Unexpectedly, male UTX-null mice escape embryonic lethality due to expression of UTY, a paralog that lacks H3K27 demethylase activity, suggesting an enzyme-independent role for UTX in development and thereby challenging the need for active H3K27 demethylation in vivo. However, the requirement for active H3K27 demethylation in stem cell-mediated tissue regeneration remains untested. Here, we employed an inducible mouse KO that specifically ablates Utx in satellite cells (SCs) and demonstrated that active H3K27 demethylation is necessary for muscle regeneration. Loss of UTX in SCs blocked myofiber regeneration in both male and female mice. Furthermore, we demonstrated that UTX mediates muscle regeneration through its H3K27 demethylase activity, as loss of demethylase activity either by chemical inhibition or knock-in of demethylase-dead UTX resulted in defective muscle repair. Mechanistically, dissection of the muscle regenerative process revealed that the demethylase activity of UTX is required for expression of the transcription factor myogenin, which in turn drives differentiation of muscle progenitors. Thus, we have identified a critical role for the enzymatic activity of UTX in activating muscle-specific gene expression during myofiber regeneration and have revealed a physiological role for active H3K27 demethylation in vivo.

Super-enhancers (SEs) are a class of compound regulatory elements which control expression of key cell-identity genes. It remains unclear whether they are simply clusters of independent classical enhancer elements or whether SEs manifest emergent properties and should therefore be considered as a distinct class of element. Here, using synthetic biology and genome editing, we engineered the well characterised erythroid α-globin SE at the endogenous α-globin locus, removing all SE constituents in a mouse embryonic stem cell-line, to create a "blank canvass". This has allowed us to re-build the SE through individual and combinatorial reinsertion of its five elements (R1, R2, R3, Rm, R4), to test the importance of each constituent's sequence and position within the locus. Each re-inserted element independently creates a region of open chromatin and binds its normal repertoire of transcription factors; however, we found a high degree of functional interdependence between the five constituents. Surprisingly, the two strongest α-globin enhancers (R1 and R2) act sub-optimally both on their own and in combination, and although the other three elements (R3, Rm and R4) exhibit no discernible enhancer activity, they each exert a major positive effect in facilitating the activity of the classical enhancers (R1 and R2). This effect depends not simply on the sequence of each elements but on their positions within the cluster. We propose that these "facilitators" are a novel form of regulatory element, important for ensuring the full activity of SEs but are distinct from conventional enhancer elements.

The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.

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