methods are described in detail in the supplementary material online , below is a brief summary . we used publicly available chip - seq data produced from the livers of human , macaque , mouse , rat , and dog for four liver - enriched tfs : cebpa , foxa1 , hnf4a , onecut1 ( schmidt et al . reads were mapped to each reference genome with bowtie2 ( langmead and salzberg 2012 ) with default parameters . numbers of mapped reads and quality metrics are shown in supplementary table s2 , supplementary material online , for the liver - enriched tfs . tf peaks were called as reproducible in both replicates if they overlapped by  75% reciprocally ( supplementary table s3 , supplementary material online ) . ( 2010 ) and were screened by eye for whether they have liver expression in mouse , rat , and dog ( based upon previously published rna - seq data , see supplementary material online ) , as well as whether they have clear 1:1 orthologs based on ensembl annotations and/or based on sequence similarity . the set of 1,373 liver expressed genes that are one - to - one orthologs across the 5 mammals was constructed based on a cut - off of  5 read fragments per kilobase of exon per million reads within the livers of mouse , rat , and dog . the binding level for orthologs across species 2015 ) . before running these analyses , for each tf and species , read alignment files were downsampled to the number of reads mapped in the replicate with the fewest number of reads . this approach combines the number of binding events , their binding intensity , and their distance from the tss into a single measure . specifically , binding level ( ais ) , where i is an orthologous gene in species s , is given by 
 ais  =  log  ( jkgijksdijks  +  0.1 ) 
 where k is a peak within 100 kb of the tss of gene i , gijks is the intensity of peak k for tf j in species s and dijks is the distance ( in bp ) from the tss to the summit of peak k. the region size used is 100  kb and is discussed further elsewhere ( wong et al . a pseudocount of 0.1 is added to the denominator to ensure this value is never zero . the mean and variance in binding level was calculated for all tfs and species was determined for all liver expressed genes . because the signal - to - noise ratio differs greatly across chip - seq experiments , binding levels were converted into standard scores ( for each species and tf ) so that they could be better compared across species . the standard scores of binding levels for each tf were then summed to obtain a single measure of binding level per gene . maximum - likelihood ancestral binding levels were reconstructed under a bm model in r using the  ace  function of the ape package ( paradis et al . enrichment levels for the histone modification markers were calculated using the same steps as for binding level . all sequence analyses reported were performed on peak and flank coordinates sliced from the 38 eutherian epo alignment ( release 74 ; cunningham et al . 2015 ) . peak sequences were taken as 100 bp from peak summits , while flanking regions were taken as 2 kb from peak ends that did not intersect other peaks or exons . these alignments were realigned with mafft ( l - ins - i method ; v6.882b ; katoh and toh 2008 ) . shared peaks across species were called based upon overlapping summits ( taken as the peak center ) within 150 bp of each other within the alignment . ancestral sequences at each node in the primate phylogeny were reconstructed using the  prequel  program of phast ( v1.3 ; hubisz et al . lrts were performed with the  phylop  program of phast to test for accelerated evolution within the human supplementary tables s1s3 , figures s1s9 , and text are available at molecular biology and evolution online ( http://www.mbe.oxfordjournals.org/ ) .
characteristics of pseudogene degeneration at the coding level are well - known , such as a shift toward neutral rates of nonsynonymous substitutions and gain of frameshift mutations . in contrast , degeneration of pseudogene transcriptional regulation is not well understood . here 
 , we test two predictions of regulatory degeneration along a pseudogenized lineage : 1 ) decreased transcription factor ( tf ) binding and 2 ) accelerated evolution in putative cis - regulatory regions.we find evidence for decreased tf binding levels nearby two primate pseudogenes compared with functional liver genes . 
 however , the majority of tf - bound sequences nearby pseudogenes do not show evidence for lineage - specific accelerated rates of evolution . 
 we conclude that decreases in tf binding level could be a marker for regulatory degeneration , while sequence degeneration in primate cis - regulatory modules may be obscured by background rates of tf binding site turnover .