Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2026 Feb 1.
Published in final edited form as: Curr Opin Genet Dev. 2024 Nov 26;90:102279. doi: 10.1016/j.gde.2024.102279

Novelty versus Innovation of Gene Regulatory Elements in Human Evolution and Disease

Anushka Katikaneni a,b, Craig B Lowe a,b,*
PMCID: PMC11769741  NIHMSID: NIHMS2038567  PMID: 39591813

Abstract

It is not currently understood how much of human evolution is due to modifying existing functional elements in the genome versus forging novel elements from non-functional DNA. Many early experiments that aimed to assign genetic changes on the human lineage to their resulting phenotypic change have focused on mutations that modify existing elements. However, a number of recent studies have highlighted the potential ease and importance of forging novel gene regulatory elements from non-functional sequence on the human lineage. In this review, we distinguish gene regulatory element novelty from innovation. We propose definitions for these terms and emphasize their importance in studying the genetic basis of human uniqueness. We discuss why the forging of novel regulatory elements may have been less emphasized during the previous decades, and why novel regulatory elements are likely to play a significant role in both human adaptation and disease.

Keywords: HAQER, HAR, Human Evolution, Novelty, Gene Regulation

Introduction

“It is currently not known whether the evolution of new morphological traits occurs largely through the modification of pre-existing cis-regulatory elements or from the generation of new elements…”

(Gompel et al., 2005)

Humans have a long-standing interest in learning about the genetic mutations that underlie traits unique to our species. For example, there has been considerable work towards understanding the relative contributions of protein-coding versus gene regulatory changes (King and Wilson, 1975; Carroll, 2003, 2005; Wray, 2007; Franchini and Pollard, 2017; Tung, 2024), as well as how many of those regulatory mutations act in cis versus trans (Song et al., 2021; Barr et al., 2023; Hansen et al., 2024). However, there has not been a focus on understanding the relative contribution to human-specific gene regulatory changes from either modifying existing regulatory elements, or forging novel elements that are unique to humans. While the field has historically focused on the former (Pollard et al., 2006b; McLean et al., 2011; Capra et al., 2013; Boyd et al., 2015; Doan et al., 2016; Girskis et al., 2021; Xue et al., 2023; Liu et al., 2024; Whalen et al., 2023), a collection of recent work has highlighted that forging novel regulatory elements is important and more favored in evolution than may have been previously thought (Mangan et al., 2022; Xiao et al., 2024; Luthra et al., 2024).

In this review, we will discuss the concept of novelty with respect to gene regulatory elements, why the forging of novel elements may have been less emphasized during the previous decades, and the importance of novel elements in human adaptation and disease.

Background

Since the chimpanzee genome was published in 2005, the field has made significant progress in cataloging the genetic changes that separate humans from the other great apes (Chimpanzee Sequencing and Analysis Consortium, 2005). However, efficiently linking these genetic changes to specific human traits has been challenging (Carroll, 2003; Varki and Altheide, 2005). This difficulty arises from the human lineage having tens of millions of mutations, most of which are neutral, or nearly neutral, and not significantly influencing a phenotype. This presents a proverbial needle-in-a-haystack problem when identifying mutations that have significantly influenced phenotypes (Varki and Altheide, 2005).

A long-standing hypothesis has been that changes in gene regulation have made significant contributions to unique human traits (King and Wilson, 1975; Carroll, 2003, 2005; Wray, 2007; Franchini and Pollard, 2017; Tung, 2024). However, testing this insight was challenging at the time due to the inefficiency in identifying gene regulatory sequences and determining whether human-specific changes in them had functional consequences (Carroll, 2003). Early publications identified human-specific changes in promoter regions. This led to exciting discoveries about the regulation of individual genes (Rockman et al., 2003; Hahn et al., 2004) as well as groups of genes involved in particular biological functions, such as glucose metabolism and neurogenesis (Haygood et al., 2007).

An insight that allowed the inclusion of distal regulatory regions was to identify human-specific mutations in otherwise conserved regions (Pollard et al., 2006a,b). It was insightful to use cross-species conservation because genome-wide methods to detect regulatory elements through open chromatin, epigenetic modifications, or protein-binding were still in their infancy, or did not exist yet (Johnson et al., 2007; Mikkelsen et al., 2007; Boyle et al., 2008). The ENCODE Project’s pilot paper, studying only 1% of the genome, was published during the following year (ENCODE Project Consortium et al., 2007). The original study describing these otherwise conserved regions with significant divergence on the human lineage termed them Human Accelerated Regions (HARs) (Pollard et al., 2006a,b). This initial screen was followed by a number of additional studies that expanded our understanding of human-specific gene regulation through changes in (Prabhakar et al., 2006; Bird et al., 2007; Boyd et al., 2015), including the deletion of (McLean et al., 2011), conserved elements. This family of screens, due to their restriction to ancestrally-conserved regions, represents pre-existing gene regulatory elements that have been significantly modified on the human lineage. Elements identified in these comparative genomic screens have been the focus of functional experiments going from genetic changes to gene regulatory and tissue-level phenotypes (Capra et al., 2013; Doan et al., 2016; Girskis et al., 2021; Xue et al., 2023; Liu et al., 2024; Whalen et al., 2023). In contrast, there has been limited experimental analysis to understand the contribution of novel gene regulatory elements to human-specific traits. This highlights an opportunity for the field to explore the role of novel regulatory elements in human evolution, as recent studies suggest their potential significance (Mangan et al., 2022; Xiao et al., 2024; Luthra et al., 2024).

The importance of defining novelty for gene regulatory elements

Researchers have proposed various definitions of biological novelty and the topic is still being actively explored. Determining a universal definition is challenging because it often needs to be tailored to the perspective and specific scale of the question being asked (DiFrisco et al., 2023; Love, 2024). For instance, a novel morphological trait has previously been defined as a structure which does not show homology with any ancestral structures or other structures in the same organism (Love, 2024). However, traits that appear novel at the level of whole-organism morphology, can be homologous to ancestral states when viewed at the level of cell types or developmental gene expression patterns (Shubin et al., 2009; Wagner, 2015). This makes it important to consider the scale at which we want to investigate novelty, that is, consider separately the novelty of cell types involved versus the novelty of morphological structures involved. While much of the work on novelty has focused on scales ranging from protein-coding genes to organismal morphology, there has been less emphasis on defining novelty at the level of gene regulatory elements.

Historically, regulatory elements have been described as novel in various contexts. This includes elements that were either newly discovered or responsible for expression in cells or tissues that did not have an ancestral expression pattern. While this derived expression pattern could be due to a newly-functional regulatory element, it could also be due to mutations in an existing regulatory element, or through the co-option of existing regulatory sequences, such as transposable elements. The novelty in expression activity was often conflated with the potential novelty of the regulatory element. That is, if change in a regulatory element leads to a novel function, the regulatory element at times was described as novel. This presents a lack of clear and consistent criteria for defining the novelty of a gene regulatory element, making it difficult to understand the relative contribution of novel regulatory elements to unique human traits.

In the quest to understand what constitutes biological novelty, previous work has argued for distinguishing between novel structures and novel functions (Wagner, 2015). At the scale of gene regulatory elements in evolutionary and functional genomics, we understand “structure” to mean the DNA sequence of the element, and “function” to mean the regulatory activity of the element. Although often closely related, novel functions can arise without the presence of novel structures, and vice versa (Wagner, 2015; Wong et al., 2020). Here, we focus on the scale of regulatory element structure, using the term “novelty” for regulatory elements derived from DNA without ancestral activity, and “innovation” for regulatory elements derived from DNA with ancestral activity. The ancestral and derived states will be defined in the context of the branch being analyzed.

To further address the ambiguity of novelty in gene regulatory element identification, we present a framework for classifying novelty and innovation for gene regulatory elements (Figures 1, 2). Articulating a classifying framework with a set of definitions will allow for several goals to be addressed in the future: (1) enable the design of specific screens to directly detect and validate novel regulatory elements, (2) allow the assessment of existing studies on regulatory element evolution within a classifying framework of definitions (Figure 1,2), and (3) facilitate the understanding of differences in regulatory evolution between humans and other species on the tree of life. Overall, a consistent framework to classify the evolution of gene regulatory elements will enhance our understanding of species-specific trait evolution.

Figure 1: Classification of regulatory element novelties.

Figure 1:

Open in a new tab

(A) Novel regulatory elements. These regulatory elements are forged from DNA not functioning in a regulatory capacity in the ancestral state. (B) Examples of mutational mechanisms that can lead to the emergence of novel regulatory elements are point mutations (left) and short indel mutations (right).

Figure 2: Classification of regulatory element innovations.

Figure 2:

Open in a new tab

(A, left to right) There are three other ways that gene regulation can be innovative without the forging of novel regulatory elements: expression domain innovation (left), regulatory association innovation (middle), and duplication associated innovation (right). (B) Regulatory domain innovation can occur through gain or change in expression domain from that of the ancestral regulatory element. This can be brought about by mutations, and lead to a change in expression levels, expression timing, or spatial location (top panel). Regulatory association innovation can occur from structural variations, leading to a change in regulatory element-target connection (middle panel). Innovation via duplication can occur from the duplication of an ancestral regulatory element through tandem, segmental, or transposon duplications.

What is a novel gene regulatory element?

We use the term “novelty” to refer to novel structures in gene regulation, where we associate structures with the regulatory element sequence itself. We classify a regulatory element as “novel” on a given lineage if it is derived entirely from bases that were ancestrally not functioning as part of a regulatory element, with this derivation occurring on the lineage being analyzed (Figure 1A). Novel elements can arise de novo through mutations in the DNA fragment (Figure 1B), or changes in the trans-regulatory environment. We consider this to be the sole way novel gene regulatory elements arise, but there are several other mechanisms that can give rise to important gene regulatory innovations.

We use the term “innovation” to refer to regulatory elements with activity different from their ancestors, which we associate with a change in the ancestral expression domain of the gene being regulated. We describe three categories illustrating how these changes lead to innovative outcomes in gene expression: (a) expression domain innovation, (b) regulatory association innovation, and (c) duplication-associated innovation (Figure 2A). Although we present regulatory novelty and innovations as distinct categories, they are not mutually exclusive and often occur in complex combinations.

  1. Expression domain innovation arises when an existing regulatory element gains an expression domain that was not part of its ancestral function (Figure 2B, top panel). This includes changes in the time or location of activity, that cause the element to be active in cells where it was not previously active. It is also innovative to change the level of expression in cells where the element was already active. An example would be an enhancer active in developing cardiac tissue gaining mutations that cause it to also be active in developing limb tissue. This expansion of expression domains likely happens frequently and may often occur by adding additional bases to the regulatory element. This expansion can lead to large and complex regulatory elements in extant genomes that drive expression in multiple domains (Gompel et al., 2005; Emera et al., 2016; Fong and Capra, 2021, 2022).

  2. Regulatory innovation through change of association arises when a regulatory element loses or gains the capacity to induce expression of a target. An example of this is “enhancer hijacking,” where a regulatory element takes control of the expression of a new target gene (Keough et al., 2023). This can occur through structural variations, such as translocations, inversions, and indels, which can impact the ancestral element by altering its genomic coordinates directly, or through changes to the neighboring DNA sequences (Figure 2B, middle).

  3. Innovation from sequence duplication arises through the increase in copy number of existing regulatory elements or the co-option of repetitive elements with known functional capacity (Figure 2B, bottom). An example would be a regulatory element that undergoes a tandem duplication (Song et al., 2018), or additional regulatory modules arising from large, segmental duplications. Many transposable element insertions giving rise to gene regulatory changes would be included in this category because they represent a complex class of repetitive DNA sequences with demonstrated regulatory capability and a history of being co-opted for gene regulation (Fueyo et al., 2022).

Novel regulatory elements may be underappreciated in human evolution

While the field has primarily focused on changes to ancestrally functional elements in humans, there are statistical frameworks that can likely enrich for detecting novel elements in computational screens. These methods have been available since the initial publishing of HARs (Siepel et al., 2006; Pertea et al., 2011; Schrider and Kern, 2015; Berrio et al., 2020), but have not received the same: attention, functional studies, or phenotypic analyses. Here, we discuss both conceptual and technical limitations that may explain why human evolutionary genetics has historically not focused experiments on novel gene regulatory elements, and why this type of evolutionary change may be underappreciated.

Evolutionary likelihood of forging novel regulatory elements

The prevailing viewpoint has been that evolving a new regulatory element from scratch is difficult and therefore would not be a favored path for adaptive gene regulatory changes (Prud’homme et al., 2007). Forging a new regulatory element from non-functional DNA would require a large number of mutations to assemble the various binding sites close to each other. Additionally, an efficient method for selection to act on intermediate steps was not apparent, with genetic changes only appearing advantageous when they are all assembled together. Tuning existing regulatory elements by strengthening or weakening binding sites, or adding or losing binding sites, would require fewer changes and allow for selection to favor intermediate steps (Prud’homme et al., 2007). In contrast, a DNA sequence with no regulatory function transforming into a regulatory element would be a substantial leap, since something complex would need to be assembled from genomic junk.

Recent work has been influential in having us reconsider this point of view, and it appears increasingly feasible to rapidly forge novel gene regulatory elements. A recent study identified a set of Human Ancestor Quickly Evolved Regions (HAQERs), which are highly diverged regions of the human genome and primarily fall outside of conserved regions. When HAQER sequences from the inferred human-chimpanzee ancestor were compared against the diverged human sequences for gene regulatory activity in neurodevelopmental cell types, many of the ancestral sequences showed activity within the range of negative controls, while the orthologous HAQER sequence acted as a strong transcriptional enhancer (Mangan et al., 2022). Additionally, recent articles have shown that many randomly generated DNA sequences have detectable gene regulatory activity in yeast (Vaishnav et al., 2022), zebrafish (Smith et al., 2013), and Drosophila (Galupa et al., 2023). This finding that some random DNA sequences regulate transcription at low levels was generalized to human cells using a machine learning approach (Luthra et al., 2024). Analyses of negative control fragments in massively parallel reporter assays (MPRAs) now incorporate the idea that a subset of randomly shuffled sequences will have a low level of gene regulatory activity (Capauto et al., 2024). From this low level of activity, there is a consensus in the field that optimization of an existing regulatory element through selection on mutational variants is efficient. The jump in thinking is that a large enough fragment of neutrally evolving DNA is likely to be constantly forging weak regulatory elements, providing a substrate that can be optimized. Forging a novel regulatory element is no longer a task of somehow assembling a number of parts needed for a functioning whole, but rather a smooth fitness surface that can be efficiently optimized by mutation and selection on intermediate states from an initial state with low activity.

This recent empirical work showing how feasible it is to forge novel regulatory elements is consistent with past theories and observations. These include an early discovery that multiple binding sites could readily evolve during a simulation of neutrally evolving DNA (Stone and Wray, 2001). Additionally, multiple groups have begun to use the term “proto-enhancer” to describe an intermediate state between non-functional DNA and what is generally considered a gene regulatory element (Emera et al., 2016; Long et al., 2016; Fong and Capra, 2021).

The theoretical ease with which novel regulatory elements can be forged predicts that there will be many species-specific differences in epigenetic states. This prediction is consistent with comparative studies discovering numerous species-specific epigenetic regulatory states in a given tissue or cell type. These studies include epigenetic signals in human limb, brain, and liver that are not observed in similar samples from Old World monkeys or more distantly related species (Cotney et al., 2013; Reilly et al., 2015; Villar et al., 2015; Li et al., 2023). Epigenetic differences have also been assayed between human and chimpanzee lymphoblastoid cell lines and brain organoids where there are many regions of open chromatin specific to human or chimpanzee samples (Shibata et al., 2012; Kanton et al., 2019; García-Pérez et al., 2021). Taken together, there is theory, observation, and experiments in model systems, and increasingly in humans, that are consistent with the forging of novel gene regulatory elements being more widespread in evolution than previously thought.

Challenges of studying novel regulatory elements

Despite the availability of statistical frameworks to detect candidate novel elements, the resulting putative gene regulatory elements are challenging to study. Investigating the functional consequences of changes in tightly conserved regions, on the other hand, offers advantages for experimental work that persist to this day. When coupled with the limited understanding of noncoding and regulatory DNA, it explains the sustained focus on these elements for functional studies over the years. Experimental studies often have a two-fold goal: identifying diverged genomic regions with potential functional consequences, and determining the functional changes imparted by these regions on the human lineage. Both of these goals may be more readily achieved in modified conserved regions.

  1. It is challenging to infer the functional significance of changes in ancestrally non-functional or unconstrained regions of the genome which may, or may not, give rise to novel regulatory elements. Many computational screens capable of detecting novel elements may have been unable to cleanly distinguish between functionally important and unimportant changes, making it difficult to assess their impact on human traits without the capability to further filter the results with high-throughput experiments Mangan et al. (2022). In contrast, genomic changes identified in tightly conserved regions are likely to have significant functional consequences. These regions, ancestrally conserved across mammals, provide strong evidence that genetic changes within them contribute to unique human phenotypes.

  2. It is challenging to experimentally determine the ancestral functions of potentially novel elements. There is less of an expectation that extant animals will contain the DNA sequence of the human-chimpanzee ancestor, or recapitulate the function of the human-chimpanzee ancestor. However, for regions constrained across mammals, DNA and tissue samples from extant mammals are more likely to accurately represent the sequence and function of the human-chimpanzee ancestor (Capra et al., 2013; Boyd et al., 2015; Girskis et al., 2021).

Despite the challenges in studying novel regulatory elements, several recent technological advancements underscore the potential for the future. Chief among these is the in vitro synthesis of large DNA fragments, allowing for direct study of the functions of inferred ancestral sequences (Klein et al., 2018; Mangan et al., 2022; Gallego Romero and Lea, 2023). Although performing experiments in the precise trans-regulatory environment of the ancestor remains difficult, recent comparative genomic studies between humans and closely related primates have begun to quantify gene regulatory activity in an evolving trans-regulatory background (Whalen et al., 2023; Hansen et al., 2024). This progress, along with other future studies, may eventually enable the study of novel regulatory elements in their ancestral trans-regulatory background. Additionally, recent studies have underscored the promise of machine learning methodologies to predict tissue-specific open chromatin as well as complex phenotypes from sequence differences (Kaplow et al., 2022, 2023). With the growing number of human and primate genomes and the development of high-throughput, parallel functional experiments (Pollen et al., 2023), there is increasing potential to develop methods and screens to understand the relative contribution of novel gene regulatory elements to human-specific traits.

Differences in the relative contribution of novel regulatory elements across species

An additional cause that may have led to a lack of focus on novel regulatory elements in human evolution is that many of the first examples of noncoding genetic changes underlying phenotypic differences were changes to ancestral regulatory elements, rather than forging of novel ones (Prud’homme et al., 2006; Rebeiz et al., 2011; Koshikawa et al., 2015). Many of these studies that worked backwards from a phenotype of interest to identify the underlying genetic changes were performed in Drosophila, which is a powerful model system for asking questions concerning the molecular basis of evolutionary change. Contributions from Drosophila research towards understanding the regulatory basis of evolution has been unparalleled. However, there may be slight differences in how flies and humans evolve.

Humans and flies may have different relative contributions to their gene regulatory changes from either modifying existing elements or forging novel ones. This is because the ratio of non-regulatory bases to regulatory bases is likely to be different across species, potentially leading to the relative contributions of forging novel regulatory elements and modifying existing regulatory elements being different. Flies, and other organisms such as yeast, have a much more compact genome than humans, with potentially 10 times more of it evolving under selective constraint (Siepel et al., 2005). Humans, and other animals such as mice, having more unconstrained sequence may lead to our species forging novel regulatory elements at a greater frequency than species with more compact genomes. This is an exciting avenue for research as we learn more about the contributions of novel elements to human evolution.

Novel Regulatory Elements and Human Disease

An important goal of studying the genetic basis of human uniqueness is to better understand human-specific diseases or diseases where humans have unique susceptibilities. If novel regulatory elements are more common than previously thought, this could significantly shift our understanding of evolutionary mechanisms in the genome and, consequently, our understanding of human disease.

If the forging of novel regulatory elements were rare, most clinically significant mutations would occur in conserved genes or regulatory regions. However, if novel regulatory elements are common in human evolution, many important regulatory elements could have been forged recently enough that disabling mutations in these regions would not be found in conserved areas. It is possible that disabling mutations in human-specific regulatory elements could be associated with the reduction of traits that we usually think of as specific to humans. Gene regulatory elements not showing cross-species conservation may be particularly important for human disease because our recent work suggests that regulatory elements may be preferentially forged in genomic regions with high mutation rates (Mangan et al., 2022). This creates an evolutionary conflict: regions with high mutation rates may generate adaptive alleles but may also produce numerous deleterious mutations within newly forged regulatory elements (Mangan et al., 2022).

If forging novel regulatory elements is very common, many human disorders could be caused not only by disrupting functional elements but also by creating novel ones that are deleterious. There is an example of this in pigs, where small mutations in non-conserved DNA upstream of the microphthalmia-associated transcription factor (MITF) gene likely forged a silencer, leading to spontaneous deafness and depigmentation (Chen et al., 2016). A potential example of this in humans is a study on congenital heart disease which found that patient-specific mutations often generated detectable regulatory element activity in regions that otherwise showed no activity (Xiao et al., 2024). Developing a deeper understanding of how regulatory elements evolve, including the relative contributions of modifications to existing elements versus the creation of novel ones, will enhance our ability to understand and treat human diseases in the future.

Future considerations

Our first consideration is how to establish a lack of function in the ancestral state, which is needed based on our definition of novelty. It will be difficult to strictly satisfy this part of the definition because it requires establishing a lack of regulatory function across all cell types, developmental stages, and environments. While advances in iPSC differentiation protocols are making rare and transient cell types more accessible, we are still far from being able to experimentally prove a lack of function across all possible cell types and conditions. This difficulty is compounded by the experiments ideally occuring not in a human or chimpanzee cell, but a cell with the trans-environment of the human-chimpanzee ancestor. It is also worth noting that many of the functional assays in this field are high-throughput reporter assays that test the function of regulatory elements outside of their native chromosomal context and with a single promoter. We are excited about future advances in iPSC differentiation and DNA synthesis, which could one day enable functional tests across a wide range of ancestral cell types and in their true ancestral genomic context.

A second consideration is whether there exists a functionless state, or if all DNA fragments may have some level of regulatory activity. Negative control DNA sequences often give a distribution of activity in gene regulatory assays rather than tightly clustering around a single value. A continuum of DNA regulatory activity across all sequences may make the forging of strong regulatory elements even more efficient, but would also require that our definition of novelty be revisited, which currently requires a non-functional state.

In this manuscript we have provided a definition of novelty for gene regulatory elements. We hope it will provide a framework for understanding the relative contribution to human-specific traits from either modifying existing regulatory elements or forging novel ones.

Acknowledgements

This publication was supported by the National Human Genome Research Institute (R35HG011332). We thank Yanting Luo for help with the figure revisions. All figures were created with BioRender.com. We gratefully acknowledge the financial support of the John Templeton Foundation (#62220). The opinions expressed in this paper are those of the authors and not those of the funders.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Declaration of interests

AK reports no conflicts of interest. CBL owns stock in Alphabet and has a family member and friends who are employees of Alphabet subsidiaries.

References

  1. Barr KA, Rhodes KL, Gilad Y: The relationship between regulatory changes in cis and trans and the evolution of gene expression in humans and chimpanzees. Genome Biol 2023. 24:207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berrio A, Haygood R, Wray GA: Identifying branch-specific positive selection throughout the regulatory genome using an appropriate proxy neutral. BMC Genomics 2020. 21:359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bird CP, Stranger BE, Liu M, Thomas DJ, Ingle CE, Beazley C, Miller W, Hurles ME, Dermitzakis ET: Fast-evolving noncoding sequences in the human genome. Genome Biol 2007. 8:R118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boyd JL, Skove SL, Rouanet JP, Pilaz LJ, Bepler T, Gordân R, Wray GA, Silver DL: Human-chimpanzee differences in a FZD8 enhancer alter cell-cycle dynamics in the developing neocortex. Curr Biol 2015. 25:772–779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boyle AP, Davis S, Shulha HP, Meltzer P, Margulies EH, Weng Z, Furey TS, Crawford GE: High-resolution mapping and characterization of open chromatin across the genome. Cell 2008. 132:311–322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Capauto D, Wang Y, Wu F, Norton S, Mariani J, Inoue F, Crawford GE, Ahituv N, Abyzov A, Vaccarino FM: Characterization of enhancer activity in early human neurodevelopment using Massively Parallel Reporter Assay (MPRA) and forebrain organoids. Sci Rep 2024. 14:3936. [DOI] [PMC free article] [PubMed] [Google Scholar]; • This study uses lentiviral vectors to perform MPRA experiments in iPSCs and forebrain organoids. The authors have made a significant contribution to the field by insightfully modeling the activity of randomly shuffled DNA sequences with a mixture of Gaussian distributions. This is consistent with an appreciable number of randomly shuffled DNA sequences having some level of gene regulatory activity.
  7. Capra JA, Erwin GD, McKinsey G, Rubenstein JLR, Pollard KS: Many human accelerated regions are developmental enhancers. Philos Trans R Soc Lond B Biol Sci 2013. 368:20130025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carroll SB: Genetics and the making of homo sapiens. Nature 2003. 422:849–857. [DOI] [PubMed] [Google Scholar]
  9. Carroll SB: Evolution at two levels: on genes and form. PLoS Biol 2005. 3:e245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen L, Guo W, Ren L, Yang M, Zhao Y, Guo Z, Yi H, Li M, Hu Y, Long X, et al. : A de novo silencer causes elimination of MITF-M expression and profound hearing loss in pigs. BMC Biol 2016. 14:52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chimpanzee Sequencing and Analysis Consortium: Initial sequence of the chimpanzee genome and comparison with the human genome. Nature 2005. 437:69–87. [DOI] [PubMed] [Google Scholar]
  12. Cotney J, Leng J, Yin J, Reilly SK, DeMare LE, Emera D, Ayoub AE, Rakic P, Noonan JP: The evolution of lineage-specific regulatory activities in the human embryonic limb. Cell 2013. 154:185–196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. DiFrisco J, Love AC, Wagner GP: The hierarchical basis of serial homology and evolutionary novelty. Journal of Morphology 2023. 284:e21531. [DOI] [PubMed] [Google Scholar]; • This paper expands on the theoretical concepts of evolutionary novelty and employs these conceptual developments to case studies at several biological levels, ranging from proteincoding genes and cell types to morphological traits such as appendages. Through these ideas, the authors address the complexity of the novelty concept while suggesting constructive ways with which it can be applied to evolutionary biology.
  14. Doan RN, Bae BI, Cubelos B, Chang C, Hossain AA, Al-Saad S, Mukaddes NM, Oner O, Al-Saffar M, Balkhy S, et al. : Mutations in human accelerated regions disrupt cognition and social behavior. Cell 2016. 167:341–354.e12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Emera D, Yin J, Reilly SK, Gockley J, Noonan JP: Origin and evolution of developmental enhancers in the mammalian neocortex. Proc Natl Acad Sci U S A 2016. 113:E2617–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. ENCODE Project Consortium, Birney E, Stamatoyannopoulos JA, Dutta A, Guigó R, Gingeras TR, Margulies EH, Weng Z, Snyder M, Dermitzakis ET, et al. : Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 2007. 447:799–816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fong SL, Capra JA: Modeling the evolutionary architectures of transcribed human enhancer sequences reveals distinct origins, functions, and associations with human trait variation. Mol Biol Evol 2021. 38:3681–3696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fong SL, Capra JA: Function and constraint in enhancer sequences with multiple evolutionary origins. Genome Biol Evol 2022. 14. [DOI] [PMC free article] [PubMed] [Google Scholar]; • This paper, along with an earlier paper from the same authors (Fong and Capra, 2021), describes a model of enhancer evolution where genomic regions can move between states of: inactivity, simple enhancer, and complex enhancer. Complex enhancers have a core region of cross-species conservation and younger flanks, which would be consistent with regulatory element novelty occuring to forge the core elements from inactive sequence, and then later regulatory innovations that refine the expression domain(s) associated with the element.
  19. Franchini LF, Pollard KS: Human evolution: the non-coding revolution. BMC Biol 2017. 15:89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fueyo R, Judd J, Feschotte C, Wysocka J: Roles of transposable elements in the regulation of mammalian transcription. Nat Rev Mol Cell Biol 2022. 23:481–497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gallego Romero I, Lea AJ: Leveraging massively parallel reporter assays for evolutionary questions. Genome Biol 2023. 24:26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Galupa R, Alvarez-Canales G, Borst NO, Fuqua T, Gandara L, Misunou N, Richter K, Alves MRP, Karumbi E, Perkins ML, et al. : Enhancer architecture and chromatin accessibility constrain phenotypic space during drosophila development. Dev Cell 2023. 58:51–62.e4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. García-Pérez R, Esteller-Cucala P, Mas G, Lobón I, Di Carlo V, Riera M, Kuhlwilm M, Navarro A, Blancher A, Di Croce L, et al. : Epigenomic profiling of primate lymphoblastoid cell lines reveals the evolutionary patterns of epigenetic activities in gene regulatory architectures. Nat Commun 2021. 12:3116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Girskis KM, Stergachis AB, DeGennaro EM, Doan RN, Qian X, Johnson MB, Wang PP, Sejourne GM, Nagy MA, Pollina EA, et al. : Rewiring of human neurodevelopmental gene regulatory programs by human accelerated regions. Neuron 2021. 109:3239–3251.e7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gompel N, Prud’homme B, Wittkopp PJ, Kassner VA, Carroll SB: Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in drosophila. Nature 2005. 433:481–487. [DOI] [PubMed] [Google Scholar]
  26. Hahn MW, Rockman MV, Soranzo N, Goldstein DB, Wray GA: Population genetic and phylogenetic evidence for positive selection on regulatory mutations at the factor VII locus in humans. Genetics 2004. 167:867–877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hansen TJ, Fong SL, Day JK, Capra JA, Hodges E: Human gene regulatory evolution is driven by the divergence of regulatory element function in both cis and trans. Cell Genom 2024. 4:100536. [DOI] [PMC free article] [PubMed] [Google Scholar]; • This study showed that there have also been significant contributions to recent gene regulatory changes from changes to the trans environment. While we envision that mutations within a fragment of DNA usually cause it to gain gene regulatory abilities, it is also possible for changes in trans-acting factors to forge a regulatory element from inactive DNA.
  28. Haygood R, Fedrigo O, Hanson B, Yokoyama KD, Wray GA: Promoter regions of many neural- and nutrition-related genes have experienced positive selection during human evolution. Nat Genet 2007. 39:1140–1144. [DOI] [PubMed] [Google Scholar]
  29. Johnson DS, Mortazavi A, Myers RM, Wold B: Genome-wide mapping of in vivo protein-DNA interactions. Science 2007. 316:1497–1502. [DOI] [PubMed] [Google Scholar]
  30. Kanton S, Boyle MJ, He Z, Santel M, Weigert A, Sanchís-Calleja F, Guijarro P, Sidow L, Fleck JS, Han D, et al. : Organoid single-cell genomic atlas uncovers human-specific features of brain development. Nature 2019. 574:418–422. [DOI] [PubMed] [Google Scholar]
  31. Kaplow IM, Lawler AJ, Schäffer DE, Srinivasan C, Sestili HH, Wirthlin ME, Phan BN, Prasad K, Brown AR, Zhang X, et al. : Relating enhancer genetic variation across mammals to complex phenotypes using machine learning. Science 2023. 380:eabm7993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kaplow IM, Schäffer DE, Wirthlin ME, Lawler AJ, Brown AR, Kleyman M, Pfenning AR: Inferring mammalian tissue-specific regulatory conservation by predicting tissue-specific differences in open chromatin. BMC Genomics 2022. 23:291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Keough KC, Whalen S, Inoue F, Przytycki PF, Fair T, Deng C, Steyert M, Ryu H, Lindblad-Toh K, Karlsson E, et al. : Three-dimensional genome rewiring in loci with human accelerated regions. Science 2023. 380:eabm1696. [DOI] [PMC free article] [PubMed] [Google Scholar]; • This study describes the enhancer hijacking of HARs where a HAR, acting as a gene regulatory element, changes its target gene. In our discussion of gene regulatory novelty, this paper is a key example of a novel regulatory association.
  34. King MC, Wilson AC: Evolution at two levels in humans and chimpanzees. Science 1975. 188:107–116. [DOI] [PubMed] [Google Scholar]
  35. Klein JC, Keith A, Agarwal V, Durham T, Shendure J: Functional characterization of enhancer evolution in the primate lineage. Genome Biol 2018. 19:99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Koshikawa S, Giorgianni MW, Vaccaro K, Kassner VA, Yoder JH, Werner T, Carroll SB: Gain of cis-regulatory activities underlies novel domains of wingless gene expression in drosophila. Proc Natl Acad Sci U S A 2015. 112:7524–7529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Li S, Hannenhalli S, Ovcharenko I: De novo human brain enhancers created by single-nucleotide mutations. Sci Adv 2023. 9:eadd2911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Liu J, Mosti F, Zhao HT, Sotelo-Fonseca JE, Escobar-Tomlienovich CF, Lollis D, Musso CM, Mao Y, Massri AJ, Doll HM, et al. : A human-specific enhancer fine-tunes radial glia potency and corticogenesis. bioRxiv 2024. [Google Scholar]
  39. Long HK, Prescott SL, Wysocka J: Ever-changing landscapes: Transcriptional enhancers in development and evolution. Cell 2016. 167:1170–1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Love AC: Evolution and Development: Conceptual Issues. Elements in the Philosophy of Biology. Cambridge University Press, 2024. [Google Scholar]; • This publication provides an examination and historical perspective on the concept of evolutionary novelties in developmental biology. It discusses the interdisciplinary and complex nature of these concepts while highlighting the importance of constituting research agendas for progress in answering biological questions.
  41. Luthra I, Jensen C, Chen XE, Salaudeen AL, Rafi AM, de Boer CG: Regulatory activity is the default DNA state in eukaryotes. Nat Struct Mol Biol 2024. 31:559–567. [DOI] [PubMed] [Google Scholar]; • This is an influential paper, which experimentally demonstrates in yeast that random DNA sequences are capable of gene regulatory activity. The authors extend this finding to humans by using a machine learning approach.
  42. Mangan RJ, Alsina FC, Mosti F, Sotelo-Fonseca JE, Snellings DA, Au EH, Carvalho J, Sathyan L, Johnson GD, Reddy TE, et al. : Adaptive sequence divergence forged new neurodevelopmental enhancers in humans. Cell 2022. 185:4587–4603.e23. [DOI] [PMC free article] [PubMed] [Google Scholar]; • A comparative genomic screen between the inferred human-chimpanzee ancestor and an extant human reference to detect the most divergent places in the human genome. The authors developed a single-cell in vivo enhancer assay to understand if the sequence divergence led to functional divergence and showed that many regions went from the ancestral version having no detectable function in the assay to the extant human allele acting as a strong enhancer.
  43. McLean CY, Reno PL, Pollen AA, Bassan AI, Capellini TD, Guenther C, Indjeian VB, Lim X, Menke DB, Schaar BT, et al. : Human-specific loss of regulatory DNA and the evolution of human-specific traits. Nature 2011. 471:216–219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W, Kim TK, Koche RP, et al. : Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 2007. 448:553–560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pertea M, Pertea GM, Salzberg SL: Detection of lineage-specific evolutionary changes among primate species. BMC Bioinformatics 2011. 12:274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pollard KS, Salama SR, King B, Kern AD, Dreszer T, Katzman S, Siepel A, Pedersen JS, Bejerano G, Baertsch R, et al. : Forces shaping the fastest evolving regions in the human genome. PLoS Genet 2006a. 2:e168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pollard KS, Salama SR, Lambert N, Lambot MA, Coppens S, Pedersen JS, Katzman S, King B, Onodera C, Siepel A, et al. : An RNA gene expressed during cortical development evolved rapidly in humans. Nature 2006b. 443:167–172. [DOI] [PubMed] [Google Scholar]
  48. Pollen AA, Kilik U, Lowe CB, Camp JG: Human-specific genetics: new tools to explore the molecular and cellular basis of human evolution. Nat Rev Genet 2023. 24:687–711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Prabhakar S, Noonan JP, Pääbo S, Rubin EM: Accelerated evolution of conserved noncoding sequences in humans. Science 2006. 314:786. [DOI] [PubMed] [Google Scholar]
  50. Prud’homme B, Gompel N, Carroll SB: Emerging principles of regulatory evolution. Proc Natl Acad Sci U S A 2007. 104 Suppl 1:8605–8612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Prud’homme B, Gompel N, Rokas A, Kassner VA, Williams TM, Yeh SD, True JR, Carroll SB: Repeated morphological evolution through cis-regulatory changes in a pleiotropic gene. Nature 2006. 440:1050–1053. [DOI] [PubMed] [Google Scholar]
  52. Rebeiz M, Jikomes N, Kassner VA, Carroll SB: Evolutionary origin of a novel gene expression pattern through co-option of the latent activities of existing regulatory sequences. Proc Natl Acad Sci U S A 2011. 108:10036–10043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Reilly SK, Yin J, Ayoub AE, Emera D, Leng J, Cotney J, Sarro R, Rakic P, Noonan JP: Evolutionary changes in promoter and enhancer activity during human corticogenesis. Science 2015. 347:1155–1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Rockman MV, Hahn MW, Soranzo N, Goldstein DB, Wray GA: Positive selection on a human-specific transcription factor binding site regulating IL4 expression. Curr Biol 2003. 13:2118–2123. [DOI] [PubMed] [Google Scholar]
  55. Schrider DR, Kern AD: Inferring selective constraint from population genomic data suggests recent regulatory turnover in the human brain. Genome Biol Evol 2015. 7:3511–3528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Shibata Y, Sheffield NC, Fedrigo O, Babbitt CC, Wortham M, Tewari AK, London D, Song L, Lee BK, Iyer VR, et al. : Extensive evolutionary changes in regulatory element activity during human origins are associated with altered gene expression and positive selection. PLoS Genet 2012. 8:e1002789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Shubin N, Tabin C, Carroll S: Deep homology and the origins of evolutionary novelty. Nature 2009. 457:818–823. [DOI] [PubMed] [Google Scholar]
  58. Siepel A, Bejerano G, Pedersen JS, Hinrichs AS, Hou M, Rosenbloom K, Clawson H, Spieth J, Hillier LW, Richards S, et al. : Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res 2005. 15:1034–1050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Siepel A, Pollard KS, Haussler D: New methods for detecting lineage-specific selection. In Apostolico A, Guerra C, Istrail S, Pevzner PA, Waterman M, editors, Research in Computational Molecular Biology. Springer Berlin Heidelberg, Berlin, Heidelberg, 2006. 190–205. [Google Scholar]
  60. Smith RP, Riesenfeld SJ, Holloway AK, Li Q, Murphy KK, Feliciano NM, Orecchia L, Oksenberg N, Pollard KS, Ahituv N: A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design. Genome Biol 2013. 14:R72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Song JHT, Grant RL, Behrens VC, Kučka M, Roberts Kingman GA, Soltys V, Chan YF, Kingsley DM: Genetic studies of human-chimpanzee divergence using stem cell fusions. Proc Natl Acad Sci U S A 2021. 118:e2117557118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Song JHT, Lowe CB, Kingsley DM: Characterization of a human-specific tandem repeat associated with bipolar disorder and schizophrenia. Am J Hum Genet 2018. 103:421–430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Stone JR, Wray GA: Rapid evolution of cis-regulatory sequences via local point mutations. Mol Biol Evol 2001. 18:1764–1770. [DOI] [PubMed] [Google Scholar]
  64. Tung J: Understanding human uniqueness in the pre-genomic era. Nat Rev Genet 2024. [DOI] [PubMed] [Google Scholar]
  65. Vaishnav ED, de Boer CG, Molinet J, Yassour M, Fan L, Adiconis X, Thompson DA, Levin JZ, Cubillos FA, Regev A: The evolution, evolvability and engineering of gene regulatory DNA. Nature 2022. 603:455–463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Varki A, Altheide TK: Comparing the human and chimpanzee genomes: searching for needles in a haystack. Genome Res 2005. 15:1746–1758. [DOI] [PubMed] [Google Scholar]
  67. Villar D, Berthelot C, Aldridge S, Rayner TF, Lukk M, Pignatelli M, Park TJ, Deaville R, Erichsen JT, Jasinska AJ, et al. : Enhancer evolution across 20 mammalian species. Cell 2015. 160:554–566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Wagner GP: Evolutionary innovations and novelties: Let us get down to business! Zoologischer Anzeiger - A Journal of Comparative Zoology 2015. 256:75–81. Special Issue: Proceedings of the 3rd International Congress on Invertebrate Morphology. [Google Scholar]
  69. Whalen S, Inoue F, Ryu H, Fair T, Markenscoff-Papadimitriou E, Keough K, Kircher M, Martin B, Alvarado B, Elor O, et al. : Machine learning dissection of human accelerated regions in primate neurodevelopment. Neuron 2023. 111:857–873.e8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Wong ES, Zheng D, Tan SZ, Bower NL, Garside V, Vanwalleghem G, Gaiti F, Scott E, Hogan BM, Kikuchi K, et al. : Deep conservation of the enhancer regulatory code in animals. Science 2020. 370:eaax8137. [DOI] [PubMed] [Google Scholar]
  71. Wray GA: The evolutionary significance of cis-regulatory mutations. Nat Rev Genet 2007. 8:206–216. [DOI] [PubMed] [Google Scholar]
  72. Xiao F, Zhang X, Morton SU, Kim SW, Fan Y, Gorham JM, Zhang H, Berkson PJ, Mazumdar N, Cao Y, et al. : Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease. Nat Genet 2024. 56:420–430. [DOI] [PMC free article] [PubMed] [Google Scholar]; • This study discovered that mutations associated with congenital heart disorders often generated regulatory element activity in an otherwise inactive DNA fragment.
  73. Xue JR, Mackay-Smith A, Mouri K, Garcia MF, Dong MX, Akers JF, Noble M, Li X, Zoonomia Consortium†, Lindblad-Toh K, et al. : The functional and evolutionary impacts of human-specific deletions in conserved elements. Science 2023. 380:eabn2253. [DOI] [PMC free article] [PubMed] [Google Scholar]; • This study identified functional consequence of deletions occuring on the human lineage in otherwise conserved regions. This is an example of gene regulatory innovation by changing the expression of an existing regulatory element.

RESOURCES