Differences in human and chimpanzee gene expression patterns define an evolving network of transcription factors in brain Katja Nowicka,b, Tim Gernata,b, Eivind Almaasc, and Lisa Stubbsa,b,1 aInstitute for Genomic Biology and bDepartment of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801; and cDepartment of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway Communicated by Gene E. Robinson, University of Illinois at Urbana-Champaign, Urbana, IL, October 4, 2009 (received for review July 7, 2009) Humans differ from other primates by marked differences in and networking of specific TFs could be driving major changes cognitive abilities and a significantly larger brain. These differences between primate species. However, previous analyses of human correlate with metabolic changes, as evidenced by the relative and chimpanzee TF gene expression did not include a compar- up-regulation of energy-related genes and metabolites in human ison of gene expression in brain. brain. While the mechanisms underlying these evolutionary Other large microarray studies have included human and changes have not been elucidated, altered activities of key tran- chimpanzee brain comparisons (5, 7, 11), but the assessment of scription factors (TFs) could play a pivotal role. To assess this TF gene expression is complicated by several issues. In partic- possibility, we analyzed microarray data from five tissues from ular, many TF loci are members of extended gene families, yet humans and chimpanzees. We identified 90 TF genes with signif- most microarray platforms are not designed to uniquely detect icantly different expression levels in human and chimpanzee brain the specific family members. The problem is particularly acute among which the rapidly evolving KRAB-zinc finger genes are for the largest family of TFs in mammals, the KRAB zinc finger markedly over-represented. The differentially expressed TFs clus- (KRAB-ZNF) genes. About one-third of these genes are pri- ter within a robust regulatory network consisting of two distinct mate-specific, including many recent duplicates (12). In striking but interlinked modules, one strongly associated with energy contrast to other TFs, KRAB-ZNFs have on average accumu- metabolism functions, and the other with transcription, vesicular lated more amino acid differences between humans and chim- transport, and ubiquitination. Our results suggest that concerted panzees than other genes, indicating that they may have con- changes in a relatively small number of interacting TFs may tributed disproportionately to the phenotypic differences coordinate major gene expression differences in human and chim- between these species (13, 14). panzee brain. To enable an accurate comparison of TF gene expression and network structure in human and chimpanzee brain, we devised comparative transcriptomics ͉ KRAB-zinc finger genes ͉ primate evolution ͉ a strategy to reliably distinguish expression levels of individual gene regulatory network evolution gene family members. Our analysis of an established dataset (11) uncovered 90 TF genes that are differentially expressed and umans differ from chimpanzees in a number of important revealed that they are organized in a coexpression network Hanatomical and physiological respects, most strikingly in our comprised of two modules with distinct functions. Both modules enhanced cognitive abilities and a substantial increase in the are enriched for primate-specific KRAB-ZNF genes, which relative size of the human brain (1). Although the human brain despite their recent advent are robustly embedded in the chim- is relatively energy-efficient per cell compared with brains of panzee and human brain networks. Our results implicate a other species, this increased capacity imposes a significant network of TFs with differential expression in human and metabolic and oxidative burden (2, 3). Several studies have noted chimpanzee brain involved in regulation of energy metabolism, the up-regulation of genes and metabolites involved in oxidative vesicle transport, and related functions required to build and metabolism and mitochondrial function in human brains com- maintain the larger and more complex human brain. pared with chimpanzee brains (2, 4, 5). These data, together with evidence of positive selection acting on the promoters of genes Results involved in energy metabolism during human evolution, indicate TF Genes with Differential Expression in Human Compared to Chim- that increased energy production has been essential to the panzee Brain. We reexamined the expression of TF genes (15) in evolution of the human brain (6). The relative up-regulation of a published Affymetrix microarray data set that contrasts five human genes in other functional categories, including neuro- human and chimpanzee tissues: heart, kidney, liver, testis, and protection and synaptic transport, has also been documented (7). brain—specifically prefrontal cortex (PFC). Although all cortex However, the molecular mechanisms underlying these well- regions display very similar expression differences between documented species differences have not been elucidated. humans and chimpanzees (16), the PFC is a good study object Although some differences in human–chimpanzee gene ex- because of its marked differences in structure and function pression may be due to cis-regulatory element divergence, between the two species. For convenience, we will refer to these transcription factors (TFs) represent another potential source of samples simply as ‘‘brain’’ in the following discussion. expression variability. Whereas most cis-element mutations would be expected to have limited, localized effects, alterations in TF sequence and/or expression could alter the expression of Author contributions: K.N. and L.S. designed research; K.N. performed research; T.G. and E.A. contributed new reagents/analytic tools; K.N., T.G., E.A., and L.S. analyzed data; and hundreds of target genes in a coordinated fashion (8, 9). Because K.N. and L.S. wrote the paper. of these predicted consequences, it is often assumed that TFs are The authors declare no conflict of interest. evolutionarily stable, and indeed, TFs as a class are structurally 1To whom correspondence should be addressed at: Institute for Genomic Biology, Univer- well conserved (8). However, two recent studies have identified sity of Illinois at Urbana-Champaign, 1206 West Gregory Drive, Urbana, IL 61801. E-mail: TF genes as enriched among genes with expression patterns that [email protected]. are under directional selection in humans (4, 10). These studies This article contains supporting information online at www.pnas.org/cgi/content/full/ raise the intriguing hypothesis that differences in the expression 0911376106/DCSupplemental. 22358–22363 ͉ PNAS ͉ December 29, 2009 ͉ vol. 106 ͉ no. 52 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0911376106 Downloaded by guest on September 24, 2021 variation within species to the interspecies difference (SI Text). On the other hand, KRAB-ZNFs are significantly depleted (PT, P ϭ 0.999) from the set of differentially expressed genes in testis. The only other large TF families, encoding Homeobox and the basic helix–loop–helix (bHLH) proteins, do not show such enrichment: Whereas 23.2% of brain-expressed KRAB-ZNFs are differentially expressed between species, only 8.2% of the Homeobox and 5.5% of the bHLH genes have changed in brain expression. Our analysis therefore revealed a clear contrast between KRAB-ZNFs and other TFs as well as between brain and other tissues (Fig. 1). To focus on the differentially expressed TFs most likely affecting brain functions, we applied three additional filters. In Fig. 1. Percentage of differentially expressed genes among human and the remainder of this paper, we will only refer to genes as chimpanzee tissues. The proportion of all genes (white), all transcription ‘‘changed’’ between humans and chimpanzees if they (i) are factors (light gray), all KRAB-ZNF (KZNF) genes (gray), conserved KRAB-ZNF significantly different in expression after correcting the P value genes (dark gray), or primate-specific KRAB-ZNF genes (black) that are differ- obtained from the two-sample t test within each tissue for entially expressed between species (t test, P Ͻ 0.01) is shown separately for multiple testing (P Ͻ 0.05; Benjamini–Hochberg correction), (ii) each tissue. Asterisks mark values that represent significant enrichment. Num- have a difference of at least 1.2-fold, and (iii) have a difference bers of genes per category are between 7 and 6,720. of 20 units of expression values. The latter criterion ensures that the gene is expressed at a modest level in at least one species. We Expression data were analyzed from all five tissues in each of found 90 TFs, including 33 KRAB-ZNFs, that met these more six individual humans and in five individual chimpanzees (11). stringent requirements for differential expression between hu- man and chimpanzee brains (Table S2). Despite the fact that To improve the reliability of the comparisons, we masked all most TFs (79 of 90) are expressed in all five tissues, about probes that do not match both genomes perfectly (10). To detect one-quarter of them (18 of 79) have changed specifically in brain. gene family members uniquely, we also removed probes with The proportion of KRAB-ZNFs that have changed only in brain more than one exact match in either genome. This approach EVOLUTION is higher (9 of 29). Interestingly, recent primate-specific KRAB-