Figure 5 .

Sevoflurane-induced alterations of TF binding maintained in sperm of F1 and F2 males. (A) K-means cluster analysis of THSSs (n = 69) present in both F1 and F2 sperm but not control or vice versa. Fold change of pair-wise comparisons (shown on the left) of 7-day–exposed F2 vs control was used in k-means clustering. The fold changes from the F1 sperm are shown on the left. The corresponding normalized subnucleosomal reads are shown on the right for control, 7-day–exposed F1, and F2 datasets. In all clusters, the alterations found in F1 sperm datasets are maintained in the F2 sperm. (B) Genome-wide distribution of THSSs present in the different clusters of (A); promoters were defined as TSS -0.5 kb. (C) Enrichment analysis of disease ontology terms associated with the 69 differential THSSs shows genes involved in mental disorders, nervous system diseases, and ASD (Comparative Toxicogenomics Database, P-value < 0.01, hypergeometric distribution and adjusted with Bonferroni correction). (D) Gene ontology of cellular component enrichment analysis associates the 69 THSSs with genes encoding proteins located at distal axons, cytoplasmic and intracellular vesicles, and synapses (PANTHER GO-SLIM, P-value < 0.01). (E) TF motif archetype enrichment analysis at differentially accessible clusters from (A) showing increased accessibility (clusters c, d, and e; left) and lost accessibility (clusters a and b; right). TF motif archetype clusters with P-value less than 0.001 based on Monte Carlo permutation tests simulated genome-wide were labeled with a pink dot. TF motif archetypes with P-value < 0.001 in the permutation tests simulated within the pool of ENCODE’s candidate cis-regulatory elements (cCREs) are marked in green. (F) Genome browser view of a 79-kb region upstream of the Stmn2 gene. A differential THSSs with the NR/20 archetype is highlighted by a blue shaded box.