Tenderness is an important indicator of meat quality. Novel isoforms associated with meat tenderness and the role of the CCCTC-binding factor (CTCF) in regulating alternative splicing to produce isoforms in sheep are largely unknown. The current project studied six sheep from two crossbred populations (Dorper × Hu × Hu, DHH and Dorper × Dorper × Hu, DDH) with divergent meat tenderness. Pooled Iso-seq data were used to annotate the sheep genomes. Then, the updated genome annotation and six RNA-seq data were combined to identify differentially expressed isoforms (DEIs) in muscles between DHH and DDH. These data were also combined with peaks detected from CTCF ChIP-seq data to investigate the regulatory role of CTCF for the alternative splicing. As a result, a total of 624 DEIs were identified between DDH and DHH. For example, isoform 7.524.18 transcribed from CAPN3 may be associated with meat tenderness. In addition, a total of 86 genes were overlapped between genes with transcribed DEIs and genes in differential peaks identified by CTCF ChIP-seq. Among these overlapped genes, ANKRD23 produces different isoforms which may be regulated by CTCF via methylation. As preliminary research, our results identified novel isoforms associated with meat tenderness and revealed the possible regulating mechanisms of alternative splicing to produce isoforms.

CCCTC-binding factor (CTCF) is a zinc-finger protein, serving an important part in the genome architecture as well as some biochemical processes. Over 70,000 CTCF binding DNA sites have been detected genome-wide, and most anchors of chromatin loops are demarcated with the CTCF binding. Various protein or RNA molecules interact with DNA-bound CTCF to conduct different biological functions, and potentially the interfaces between CTCF and its cofactors can be targets for drug development. Here we identify the effective region of CTCF in DNA recognition, which defines the exposed CTCF surface feature for the interaction of cofactors. While the zinc-finger region contributes the most in DNA association, its binding affinity varies based on different DNA sequences. To investigate the effectiveness of individual zinc-fingers, the key residues are mutated to inactivate the DNA binding ability, while the finger configuration and the spacing between fingers are preserved. The strategy is proved to be successful, while clear differences are observed in the DNA binding affinities among the 11 finger mutants and the result is consistent to previous studies in general. With the help of inactivated finger mutants, we identify the ineffective fingers and the dominant effective fingers, which form distinctive patterns on different DNA targets.