An integrative view of dynamic genomic elements influencing human brain evolution and individual neurodevelopment

Medical Hypotheses (2008) 71, 360–373

www.elsevier.com/locate/mehy

An integrative view of dynamic genomic elements influencing human brain evolution and individual neurodevelopment G.S. Gericke

*

Department of Biomedical Sciences, Tshwane University of Technology, P.O. Box 2040, Brooklyn Square 0075, Pretoria, Gauteng, South Africa Received 1 March 2008; accepted 6 March 2008

Summary An increasing number of reports of rearranged and aneuploid chromosomes in brain cells suggest an unexpected link between developmental chromosomal instability and brain genome diversity. Unstable chromosomal fragile sites (FS), endogenously or exogenously induced by replicative stressors, participate in genetic rearrangement and may be key features of epigenetically modified neuroplasticity. Certain common chromosomal FS are known to function as signals for RAG complex targets. Recombinase activation gene RAG-1 directed V(D)J recombination affecting specific recognition sequences allows the immune system to encode memories of a vast array of antigens. The finding that RAG-1 is transcribed in the central nervous system raised the consideration that immunoglobulin-like somatic DNA recombination could be involved in recognition and memory processes in brain development and function. Cognitive stress induced somatic hypermutation in neurons, similar to what happens after antigenic challenge in lymphocytes, could underly a massive increase in the synthesis of novel macromolecules to function as coded information bits which get selected for memory storage. This process may involve mobile element activation, which may also play a role in recombinational repair. As a source of tested, successful new open reading frames, somatic hypermutation may confer a selective advantage if somatically acquired information is fed back to germline V gene arrays and the human brain could have adopted a similar process to manage the information captured in rearranged sequences. In neuroevolution and individual brain development, germline information could thus represent a crucial component. The brain itself may, from an evolutionary genetic point of view, represent nothing more than a highly specialized and individually diversified information accrual and memory system to increase the overall phenotypically validated information content of the immortal germline. In the evolution of rapid evolvability, exceeding the narrow margins within which genetic instability is useful, would be expected to be associated with penalties in terms of neuropathology and malignancy risk. The utilisation of genetic instability to obtain diversification under stress is an ancient principle, but may have reached unprecedented levels in humans, which, in turn, fed back to creation of more unstable environments. Since increased genomic instability is likely to have been introduced to the genomes of other life forms by some of the extremely genotoxic environments created by H sapiens, a better understanding of the

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0306-9877/$ - see front matter c 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2008.03.048

An integrative view of dynamic genomic elements influencing human brain evolution

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implications of borderline genomic instability may be an important priority to ensure long term biological survival, including that of our own species. c 2008 Elsevier Ltd. All rights reserved.



General evolutionary background Much speculation has centered around indications of some profound change(s) about one hundred thousand years ago which subsequently led to the accelerated divergence of modern humans away from other lineages closest to us. Independently of this consideration, a large number of models have been developed to understand various general aspects of the evolution of mutation rates and genome rearrangement since the pioneering studies of Alfred Sturtevant in the 1930s, and later Barbara McClintock who received a Nobel Prize in 1985 [1,2]. The evolvability of mutation rate plasticity involves mechanisms that eventually generate more rapid cooperative and nonlethal functional variation on which Darwinian selection can operate [3]. More recently it has been restated [4] that our concept of a stable genome is evolving to one in which genomes are plastic and responsive to environmental changes. Growing evidence shows that a variety of environmental stresses induce genomic instability in bacteria, yeast, and human cancer cells, generating occasional fitter mutants and potentially accelerating adaptive evolution [4]. The restriction of random mutagenesis in genomic space is considered to be possible if achieved via coupling of mutation-generating machinery to local events such as DNA-break repair or transcription. Such localization is considered to have the potential to minimize accumulation of deleterious mutations in the genomes of rare fitter mutants, and promote local concerted evolution [4]. The suggestion is made in the current article that either novel external stress factors and/or concurrent upregulated sensitivity to stress induced genomic rearrangement were associated with increased, controlled instability in brain and germline genetic information management structures underlying the divergence of the human lineage. Large-scale genomic rearrangements, including insertions, deletions, translocations and duplications have been recognised as potential contributors to major genetic differences between humans and primates [5]. The changes leading to modern humans are proposed to have involved specific DNA structural and sequence elements, along with increased small world network connectivity of regulatory cis-acting elements, and could have accelerated the divergence process phenomenally in terms of ordinary

evolutionary timescales. All of these are speculated to have allowed a stronger continuum of information management between genes and the environment, also including an expansion of brain– germline cooperation to store or transmit sequence coding information between generations. Comparison of fine-scale recombination rates at orthologous loci in humans and chimpanzees showed that, despite approximately 99% identity at the level of DNA sequence, recombination hotspots were found rarely (if at all) at the same positions and that recombination rates in humans have evolved rapidly in a manner disproportionate to the change in DNA sequence [6]. An analysis of human and chimpanzee sequence data indicated that protein evolution was more than 2.2 times faster in chromosomes that had undergone structural rearrangements [7]. For instance, FOXP2 is a transcription factor involved in speech and language development. Language as a grammatical system appeared perhaps as recently as 50,000 years ago and seems to be exclusive to Homo sapiens [8]. Human FOXP2 experienced a >60-fold increase in substitution rate and incorporated two fixed amino acid changes in a broadly defined transcription suppression domain. A survey of a diverse group of placental mammals revealed the uniqueness of the human FOXP2 sequence and a population genetic analysis indicated possible adaptive selection behind the accelerated evolution [9].

Specific DNA breakpoints appear to be implicated In a comparative primate study more than 80% of evolutionary breakpoints have been associated with specific chromosomal fragile sites [10] that are normally stable in somatic cells but predispose to intrachromosomal recombination and DNA strand breakage under conditions of replicative stress [11]. Biochemically inducible ‘common’ chromosomal fragile sites (CFS) are found in all individuals, extend over large regions and are associated with transcriptional activity [12]. Several of the currently known human CFS regions span large genes that extend from 700 kb to over 1.5 Mb of genomic sequence. Many of these genes have been functionally linked with neurological development [13]. Epigenetic maintenance of gene expression requires that promoters are maintained free of

362 nucleosomes [14]. Analysis of the DNA structural characteristics showed that CFS contained more areas of high torsional flexibility with more ATdinucleotide-rich islands than neighbouring nonfragile regions [15], and the decrease in the efficiency of nucleosome assembly observed in AT-rich minisatellite repeats, result in the decondensation defects seen as CFS. Short, polymorphic AT-rich regions in palindromic structures increase chromosome breakage by forming secondary structures that stall replication fork progression [16]. The main physiological source of DNA strand breaks in all cell types is due to collapsed replication forks [17], but in vivo, following a replication block, rare cells could escape checkpoint mechanisms and enter mitosis with altered genome assembly [18]. In bacteria, the key priority of unstable genetic systems during stress is to ensure survival. Therefore, the repair of lethal DNA lesions is an absolute necessity, while perfect restoration of original genetic information is not. Furthermore, the nature of DNA lesions might render error-free repair too costly, or even impossible for stressed cells [19]. Error prone repair by DNA polymerase eta targets A–T pairs [20], of which there is an abundance associated with CFS. This may be an important normal diversifying mechanism below a certain threshold. The G2/M checkpoint, which prevents entry into mitosis, has recently been shown to have a defined threshold of 10–20 double strand breaks (DSBs) [21], which could possibly allow the creation of genomic diversity at low instability levels as a normal component of ‘genomic plasticity’.

Palindrome characteristics It has been suggested that tandem repeats appear as a result of D-loop formation when singlestranded DNA forms a hairpin structure in palindromes (inverted repeat structures). Palindromes represent a range of interspersed repetitive DNA sequences capable of forming a variety of unusual DNA structures [22]. The coding capability of tandem repetitive DNA sequences operates on several levels. Most of these DNA codes are not exclusively based on the primary DNA sequence itself, but also seem to include specific features of the corresponding higher order structures [23]. The localized sequence of DNA determines the physical properties of a stretch of DNA, and that in turn determines the opening profile of that DNA fragment. Predicted openings correlate with promoter transcriptional start sites, major regulatory sites and transcriptional activity [24]. Palindromes have been considered important in the evolutionary remodelling of the genome [25] and the repeat

Gericke number is a critical parameter that helps maintain a balance between the advantage gained from an unusual structure during gene expression and the disadvantage posed by the same structure during replication [26]. In yeast, genomic instabilities caused by inverted DNA repeats located as far as 21 kb from each other lead to several chromosome rearrangements in response to a single DSB. The DSB initiates a pairing interaction between inverted repeats, resulting in the formation of large dicentric inverted dimers. Propagation of cells containing inverted dimers led to gross chromosomal rearrangements, including translocations, truncations, and amplifications [27], as DSBs in the center of the palindrome are associated with illegitimate recombination events between similar AT-rich sequences [28]. Rearrangements that maintain the central symmetry continue to be unstable. Asymmetric deletions are stabilised so that they cannot undergo further illegitimate rearrangements [29]. The formation of stable structures offers a mechanism of unwinding which is advantageous during transcription. These unusual DNA structures also provide unique’protein recognition motifs’ quite different from a Watson–Crick double helix [26].

CFS, network formation and domain boundary specification When data on CFS expression were analysed in a network context, it appeared that fragility is linked to a coordinated regulation of fragile gene expression, signaling an unexpected large connected component [30]. AT islands in CFS have been shown to function as nuclear matrix attachment regions (MARs) both in vitro and in vivo [31], which constitute the functional coordinate system for genomic regulatory regions [32]. DNA duplexes of AT islands are prone to base unpairing due to their unusual flexibility characteristics, which are necessary MAR attributes. Recent studies on the molecular mechanisms involved show that proteins of the nuclear envelope participate in regulation of transcription on several levels, from direct binding to transcription factors to induction of epigenetic histone modifications [33]. MARs organize chromosomal loops in the interphase nucleus, are about 200 bp long, AT-rich, contain topoisomerase II consensus sequences and other AT-rich sequence motifs; often reside near cis-acting regulatory sequences, and their binding sites are abundant (greater than 10,000 per mammalian nucleus) [34]. The regulatory genome supplies an enormous computational capability with the capacity to process in parallel a vast number of regulatory inputs,

An integrative view of dynamic genomic elements influencing human brain evolution comprising many thousands of processing units in the form of cis-regulatory modules that create a network [35]. Cis-elements can be defined to include the repeat sequence units, the length and purity of the repeat tracts, the sequences flanking the repeat, as well as the surrounding epigenetic environment, including DNA methylation and chromatin structure [36]. Differential network connectivity identify species-specific network connections as key drivers of evolutionary change [37]. Contacts between cis-acting sequences through the formation of chromatin loops form the most basic level of organization that impedes or permits access of factors to the genes [38]. A complex behavioural phenotype is considered to manifest itself as an emergent property of such networks [39]. Duplicate genes rapidly diverge in their expression profiles in the network and contribute to maintaining network robustness as compared with singletons [40] and according to modelling analyses, duplication plays an important role in feed-forward loop evolution [41]. Gene copy number variation has been considered to underlie a significant proportion of normal human variation including differences in cognitive, behavioural, and psychological features [42]. Analyses support a nonrandom model of chromosomal evolution associated with both recurrent small-scale duplication and large-scale evolutionary rearrangements [43]. Similarly, the human brain appears to have developed anatomically by the divergent modification of pre-existing parts [44] and new areas may have evolved as a result of processes likely to be linked with underlying extensive duplication of transcription factors [45] or genes.

Chromosomal instability in differentiating and mature neurons Findings of rearranged and aneuploid chromosomes in brain cells suggest a link between developmental chromosomal instability and brain genome diversity [46,47]. Examination of metaphase chromosome constitutions of cortical neurons in adult mice, visualized by a nuclear transfer technique, showed that although some reconstructed oocytes cloned from neuronal nuclei have an apparently normal karyotype, the majority do not, supporting a hypothesis that fully differentiated neurons in adult mammalian brains are genomically different from somatic cells [48]. In humans, previously unrecognized large-scale double-stranded DNA breaks are now known to occur under normal circumstances in early postmitotic and differentiating neurons [49]. In general, accumulation of DNA breaks in differentiating cells cannot be attributed

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to a decrease in the DNA repair efficiency. It has been hypothesized that DNA fragmentation is an epigenetic tool for regulating the differentiation process [50]. For example, CFS FRA13A breaks are limited to a 650 kb region within the neurobeachin gene, which genomically spans approximately 730 kb. Neurobeachin encodes a neuron-specific multidomain protein implicated in membrane trafficking that is predominantly expressed in the brain [51].

Chromosomal ploidy variation in normal brain Not only breakage and rearrangement and associated structural sequelae, but also large-scale chromosomal ploidy alterations seem to have been recruited as a diversifying process. Metaphase chromosome spreads from whole brains of the teleost Apteronotus leptorhynchus revealed an euploid complement of 22 chromosomes in only 22% of the cells examined. Together with the recent discovery of aneuploidy of both neurons and nonneuronal cells in the adult mammalian brain, investigations suggest that the loss or gain of chromosomes might provide a mechanism to regulate gene expression in the adult vertebrate brain [52,53]. One possible consequence of nervous system aneuploidy is altered gene expression through loss of heterozygosity [54]. Aneuploid neurons were found to be functionally active and demonstrate that functioning neurons with aneuploid genomes form genetically mosaic neural circuitries [55]. The average aneuploidy frequency has been found to be 1.25–1.45% per chromosome, with the overall percentage of aneuploidy tending to approach 30–35%. Furthermore, such mosaic aneuploidy appears to be exclusively confined to the brain [46], and it is probably crucial to contain the extensive rearrangement processes in brain cells in order to prevent this extent of breakage and ploidy alterations from creating havoc in other mitotically active cells.

Recombinase activation gene RAG-1 activity in the brain The view has been expressed that natural selection has the effect of integrating systems for the transmission of genetic information with systems relating to the external adaptation of the organism [56]. A broader repertoire of human emotions further diversified stress-associated information management. Stress hormones participate in modulation of memory consolidation processes in both the amygdala and the hippocampus [57]. Steroid

364 receptors function by binding to specific structural elements in the regulatory regions of target genes by recruitment of cofactors that modify histones and chromatin structure [58]. The emerging idea is that lifelong behavioural memory storage may involve lasting changes in the physical, three-dimensional structure of DNA itself [59,60]. The immunological recombination system has been considered before as a significant evolutionary force not limited to rearrangements only at antigen-receptor loci [61]. Recombinase activation gene RAG-1 directed V(D)J recombination is limited to specific recognition sequences that allows the immune system to encode memories of a vast array of antigens [62] and lymphoid cells purposely introduce DNA DSBs into their genome to maximize the diversity and effector functions of their antigenreceptor genes [63]. The finding that RAG-1 is transcribed in the central nervous system [64] raised interest that immunoglobulin-like somatic DNA recombination could be involved in developmental programming as well as genomic structural plasticity underlying recognition and memory processes in brain development and function [65]. Other supportive findings for the importance of RAG processes in the central nervous system include: (a) DNA rearrangement activity increases during neural differentiation [66]. (b) Maternally derived Ig light chain in the fetal murine nervous system implicates Ig molecules as potential mediators of cortical developmental events [67]. (c) The expression of RAG-1 in mouse embryonic brain tissue is higher than in the adult mouse [68]. The RAG-1 gene has been localized to neurons in the hippocampal formation and related limbic regions that are involved in spatial learning and memory [69]. Research findings provide a formal demonstration that certain CFS can function as signals for RAG complex targets [70]. Conversely, CFS were found to be enriched for genes associated with the immune response [30]. RAG proteins have been proposed to contribute to chromosomal translocations in general [71], suggesting that these may be involved in immune-like stress induced rearrangement processes following breakage at CFS. Evidence of the importance of DNA rearrangement in essential neurogenic processes also highlighted recent discoveries of genes encoding neuronal adhesion protocadherins which display structural similarity to immunoglobulins. Both the immune system and the brain evolved from a cell adhesion system [72]. Chromosomal alterations in the nucleus of differentiated neurons have been linked with cadherin-related neuronal receptor/protocadherin transcript variance [73], some of which are specific to the hominoid lineage [74].

Gericke

RAG, CFS and mobile elements Barbara McClintock originally proposed that mobile elements restructure host genomes as an adaptive response to environmental challenge [75]. Retroelements establish and refine target gene networks of transcription factors in a species-specific manner [76]. It has been suggested that the RNA regulatory network of higher eukaryotes can re-wire itself, and employ various and evolvable mutational strategies in response to external pressures enabling intracellular, RNA-mediated learning processes. Successful strategies and pathways are then recorded (hard-wired) into the DNA genome via reverse transcriptase [77]. Adenosine to inosine RNA editing, catalyzed by Adenosine Deaminases Acting on RNA (ADARs), represents an evolutionary conserved post-transcriptional mechanism which harnesses RNA structures to produce proteins that are not literally encoded in the genome. ADARs have been shown to regulate neuronal gene expression through a remarkable variety of disparate processes. The unshackling of the proteome from the constraints of the genome through RNA editing may have been fundamental to the evolution of complex behaviour [78]. The distance and homology between palindromic inverted repeats correlate with editing levels of Alu-containing mRNAs [79]. Alu family sequences are middle repetitive short interspersed elements (SINEs) dispersed throughout vertebrate genomes that can modulate gene transcription, with up to 75% of all known genes having Alu insertions within their introns and/or UTRs [78]. RAG proteins are able to capture exogenous target DNA molecules and carry out authentic transposition of signal ends into these targets [17]. Hairpin DNA structures formed in palindromes are intermediates in V(D)J recombination [29] and are formed by a chemical mechanism very similar to the early steps of transpositional recombination and retroviral integration [80]. Altered sequences arising from chromosomal rearrangement and associated TE upregulation during ‘cognitive stress’ may result in neurospecific immune-like sequelae involving CFS as key participating regions. It has been claimed that the process of memory consolidation in the brain involves the synthesis of novel macromolecules recognized by the immune system as’’non-self’’ antigens [60]. Immune-like stress induced somatic hypermutation in neurons could be similar to what happens in lymphocytes and may underly a massive increase in the synthesis of novel macromolecules to function as coded information bits following a cognitive challenge. It has been proposed that extensive genome

An integrative view of dynamic genomic elements influencing human brain evolution repatterning can occur during transient stress phases during which some epigenetic features, such as DNA methylation, are relaxed, thus allowing transposable element (TE) amplification [81]. The most common sequence feature of characterized in vivo insertion sites is that they are AT-rich [82] and a number of studies have shown a statistical association between the integration of transforming DNA viruses and expression of CFS following DNA strand breakage [83]. Analysis of genomic rearrangement breakpoint regions has revealed specific transposable element (TE) repeat density patterns, suggesting that TEs may have played a significant role in chromosome evolution and genome plasticity [84]. Oxidative stress is a determinant of long interspersed nuclear element-1 activation [85] and also induces chromosomal breaks, which may indicate some functional relationship between these phenomena [86]. LINE-1 retrotransposons, which constitute approximately 10% of the mouse genome, were present at 25% of breakpoints examined, suggesting their participation in rearrangements [87].

Memory maintenance and disassembly It has been demonstrated in rodents that the stability of DNA methylation in postmitotic cells provides a possible molecular scaffold by which changes in gene expression and behavioural traits induced by postnatal maternal care are maintained throughout life [88]. DNA methylation as a stable, but not irreversible epigenetic signal, has been implicated in brain function and the development of the immune system [89] and together with histone modificationmediated gene regulation are considered crucial for high-order cognitive functions such as learning and memory [90]. Hypomethylation of the genome largely affects the intergenic and intronic regions of the DNA, particularly repeat sequences and TEs, and is associated with chromosomal instability [81], promoting structural rearrangement. Sustained epigenetic mechanisms of gene regulation in mature neurons is supported by the observation that the histones of nonproliferating, terminally differentiated cells undergo continuous replacement [91]. Despite postmitotic neurons remaining in permanent mitotic quiescence, they also express a number of cell cycle regulators [92]. Some of these may have additional functions in terms of genome integrity management and apoptosis signaling. The suppression of cell cycle activation is considered neuroprotective [93] as it may shield information storage in altered genetic structures. Recent findings showed that key cell cycle checkpoint genes are important for genome stability at

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CFS and some observations can be interpreted to indicate that subjective memory could be affected by structural damage to rearranged DNA structures. In Alzheimer disorder (AD), cell cycle activation could theoretically disrupt 3-D genomic memory storage structures as presenilin-1 mutations linked to familial AD lead to accumulation of cyclin D1 and neuronal apoptosis secondary to an abortive entry into the cell cycle [94]. Recent studies show elevated reactive oxygen species (ROS) products in both nuclear and mtDNA isolated from vulnerable brain regions in amnestic mild cognitive impairment [95], as well as in AD [96]. Small amounts of ROS act as physiological messenger molecules in cell signal transduction pathways [97]. Neuronal membranes contain a high proportion of polyunsaturated fatty acid and is also the site for oxidative stress [98] with ROS upregulation. MDMA (‘Ecstasy’), at doses comparable with those self-administered by humans, produces acute oxidative stress and DNA DSBs, which persist together with long-lasting metabolic changes in the hippocampal formation. Persisting effects include cognitive impairment, reduced seizure threshold, long-lasting slowing of EEG activity, and hyperexcitability of the hippocampus [99]. Results of a comparative genetic analysis of chromosomal aberrations and the state of interphase chromatin in nerve cells from the developing brains of rat embryos from lines with nervous systems of different excitation thresholds showed a relationship between the basal level of nervous system excitability and cytogenetic characteristics [100]. These observations suggest a possible link between EEG characteristics, altered chromosome architecture, ROS-induced damage and modified gene expression patterns.

Structural instability, neuropathology and cancer Available data support the hypothesis that chromosomal breakage in pathological disorders non-randomly concentrate in those evolutionary chromosome bands which correspond to CFS and/ or intrachromosomal telomeric-like sequences [101]. Perhaps one differentiating characteristic of the human brain is that its exploitation of genomic rearranging and diversifying repair principles has been taken so far from equilibrium that the risk for pathological genomic instability increased substantially. Could runaway instability contribute to disturbing prevalence trends of some neurodevelopmental disorders? One particular study revealed that, just between 1989 and 2000, substance-related disorders increased by 39% and affective

366 disorder increased by 138%. Rates of autism and ADHD nearly quadrupled over the course of the study [102]. In schizophrenia, a condition with a constant worldwide prevalence, increased chromosomal fragility has been documented along with altered cell cycle control and apoptosis [103,104]. Furthermore, antipsychotics produce complex effects on apoptotic regulation in the central nervous system, activating both proapoptotic and antiapoptotic signaling pathways [104]. Yet, whatever underlies schizophrenia-associated genetic instability and altered cell cycle- and apoptotic characteristics, appears to include a mechanism protective against malignancy. At least six published incidence studies of the relative risk of cancer in patients with schizophrenia compared with the general population concluded that schizophrenia is associated with a lower risk of developing cancer [105]. An improved understanding is required of the critical determining factors distinguishing evolutionary and neurodevelopmental phenotypic diversifying mechanisms from neuropathological and malignancy associated genomic instability.

Genomic instability and cancer An instability/cancer relationship is manifested by the observations regarding chromosome rearrangements in cancer [106]. Does cancer associated with genomic instability represent the penalty we pay for having evolved a complex brain? The similarities with mechanisms involved with brain chromosome rearrangements have led to the suggestion that there are important potential linkages between normal neurological development and the development of cancer mediated by alterations in CFS genes [13]. In a process rather similar to what is proposed in this paper as normal neurological phenomena, tumour cell populations change in response to certain stresses and harbour populations of cells with increasingly diverse abilities (‘tumour cell heterogeneity’). Tumour cell heterogeneity arises as a result of genetic instability, by which the genetic material cell undergoes changes in structure, conformation and function [107]. CFS function as tumour suppressors but are not traditional mutational targets in cancer [13]. Many CFS regions contain tumour suppressor genes which appear to indicate the necessity for control mechanisms regulating their diversifying signal properties in normal (neuro)development. Epigenetic silencing of tumour suppressor genes has been shown to involve heritable changes in nucleosome occupancy [108]. Two breakpoint clusters at FRA3B have been shown to form phased nucleosomes

Gericke [109]. Though altered FRA3B CFS expression have been associated with certain cancers, results from a recent study suggested that the Fhit protein encoded by the DNA damage-susceptible FRA3B/FHIT region is normally responsible for protecting cells from accumulation of DNA damage through modulation of checkpoint proteins [110]. Chromosomes with FRA3B deletions exhibited significantly decreased fragility of this locus compared with controls [111] which may indicate some selection process accompanying tumour suppressor loss. Normal transcriptionally related CFS expression may therefore differ fundamentally from malignant transformation with altered CFS gene expression modified through intralocus deletions. Responses to RAG-specific DNA double strand breaks have been demonstrated to suppress cancer [112], for instance CpG methylation protects karyotypic stability in cells with increased V(D)J recombination [113]. Unique immunoglobulin sequence motifs or secondary structures ensure that only V(D)J sequences mutate whilst other regions of the genome are not affected [114]. DNA DSBs normally induce a signal transmitted by the ataxia-telangiectasia mutated (ATM) kinase, which suppresses illegitimate joining of DSBs and activates cell cycle checkpoints. Silencing this checkpoint permits DNA ends produced by V(D)J recombination in a lymphoid precursor to serve as substrates for translocations with chromosomes subsequently modified by other means in mature cells [115]. ATM functions directly in the repair of chromosomal DNA DSBs by maintaining DNA ends in repair complexes generated during lymphocyte antigenreceptor gene assembly [116].

Brain and germline links In humans, a prolonged period of neurodevelopmental plasticity before reproduction may have enhanced mechanisms allowing stress induced molecular memory to benefit future phenotypes. This would likely be based on RNA/epigenetic mechanisms so it could transmitted but also be erased or stored and/or retrotranscribed when necessary. Molecular memory marks for active transcription are coordinated with cell cycle events, such as replication, mitosis and nuclear organization, to mediate transcription memory across cell division events [117], and this principle may even operate intergenerationally if germline genetic information is somehow influenced by changes in brain genetic structures. The effect would be to exponentially increase the rate at which the germline could evolve (and by implica-

An integrative view of dynamic genomic elements influencing human brain evolution tion the brain), by using an ever increasing amount of acquired, stress induced regulatory sequence information, much like the effect of compound interest in long term investments. Inheritance of acquired somatic information implies a crossing of the soma to germline barrier contrary to the largely prevailing notion, first proposed by August Weismann in 1883, to limit inheritance of genetic information to the germline. While the fundamental tenet remains true, it is, however, possible that mechanisms have evolved to transfer individual acquired brain information to germline genetic information management systems. The latter would then be expected to also play a major role in specifying the type of brain that would be needed. A survey of positively selected genes in the genomes of humans and chimpanzees indicated that genes with maximal expression in the brain show little or no evidence for positive selection, while genes with maximal expression in the testis tend to be enriched with positively selected genes. Many of the genes that present a signature of positive selection in the evolution of the human lineage are involved in sensory perception, immune defenses, tumour suppression, apoptosis and genes involved in spermatogenesis [118]. Recent brain evolution may thus have accelerated through significant network modification in the germline. All of this may be aimed to ensure strong competitive selection for brain modifying genes. Due to a demonstrated higher mutation rate in the male germline, potentially useful novel sequences could originate in the testes, which would require ‘testing’ in a phenotype, the main aim being the diversification of the germline. As an example, germline human endogenous retroviruses (HERVs) appear to have played a significant role in the evolution and divergence of Hominoidea superfamily [119]. Proteins encoded by the HERV-W multicopy gene family are normally expressed in cells of the central nervous system and have been suggested to have a physiological function in the human brain [120]. The brain might therefore represent nothing more than a sophisticated phenotypic tool to enrich germline information. For instance, comparative gene mapping and sequencing shows that the male sex determining SRY gene arose quite recently as a degraded version of the SOX3 gene on the X chromosome. SOX3 is expressed predominantly in brain, and so is more likely to be a brain-determining than a testis-determining gene [121].

Germline instability It has been demonstrated that complete repair is not required for completion of mammalian meiosis

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where approximately 4% of recombinant molecules display complex and incomplete repair [122]. An essential ‘minimum’ level of breakage and recombination could play an important role in uncoupling undesirable linkage which could arise as a consequence of a high frequency of genomic rearrangement. In a constantly rearranging genome, novel genetic linkages can become problematic when complete linkage develops between loci affecting fitness and loci affecting mutation rate, following which positive natural selection and recurrent mutation can drive mutation rates in an adapting population to intolerable levels [123] and cause the type of runaway sexual selection first described by Fisher in 1930 [124]. Brain and germline epigenetically sensitive rearranging regions may stem from adapted stress response mechanisms. For instance, enhanced heat shock gene expression in response to various stimuli is regulated by heat shock transcription factors (HSFs). Certain specialized functions of HSFs include regulation of novel target genes in response to distinct nonstressful stimuli often associated with redox-dependent activation and epigenetic gene regulation [125]. Recent evidence has been found for both cooperative and specific functions of HSFs that expand beyond the heat shock response including neural plate induction in early mammalian CNS, brain development [126] and maintenance of sperm production [127]. Additional findings underscore the importance of linkage of germline and somatic genomic rearranging events [128], the identification of translin (testis-brain RNA-binding protein) recognition sites at breakpoints [129] and the finding that DNA-damaging reagents initiate a signaling pathway for the active nuclear transport of translin, support the hypothesis that translin has a pivotal function in recognition of the generated single-stranded DNA ends following staggered breaks at recombination hot spots [130]. Translin is a widely expressed and highly conserved protein with proposed functions in chromosomal translocations, mitotic cell division, and mRNA transport and storage in mouse male germ cells and neurons, also found in humans. Homozygous translin knock-out mice demonstrate altered expression of multiple mRNA transcripts in the brain and exhibit sex-specific differences in tests of learning and memory, locomotor activity, anxiety-related behaviour, and sensorimotor gating, as well as handling-induced seizures and alterations in monoamine neurotransmitter levels in several forebrain regions. These observations were considered to indicate that mutations in translin could contribute to certain human neurobehavioural disorders [131]. In adult flies, Drosophila translin

368 is localized in the brain and in early spermatocytes. Drosophila translin mutants exhibit a sex-specific impaired motor response. Drosophila translin is suggested to possess roles in neuronal development and behaviour analogous to that of mouse translin [132], indicating that a brain–gonadal link is a universal feature not restricted to mammals.

Germline V gene families The germline V gene families appear to have evolved to exploit the diversity created by somatic hypermutation [134], and the possibility needs to be investigated whether the brain adopted this immunological process to manage individual cognitively modified sequence information, including feedback to the germline. In humans, the germline contains a tandem array of highly homologous variable (V) gene elements which encode part of the antigen-binding region of the antibody protein. Germline V elements by themselves are never transcribed or translated but are only expressed in mature lymphocytes (and neurons?) following translocation on a somatic chromosome to give a typical V(D)J rearrangement. Analysis of germline V gene sequences shows that major recombination site characteristics are consistent with a hypothesis that over evolutionary time cDNA derived by reverse transcription of pre-mRNA in B lymphocytes has recombined with germline DNA [135,136]. In terms of the need for immune diversity, somatic hypermutation by itself now appears to be irrelevant as there is said to be sufficient germline diversity and combinatorial diversity of genetic elements encoding heavy and light chains of antibodies [62]. However the feedback to the germline of tested successful new open reading frames may confer a selective advantage by reducing the effect of random genetic drift which could potentially degrade the germline V repertoire.

Palindrome arm gene conversion Retroduplicated gene transcripts are found most abundantly in testes and retroduplications show a higher level of nucleotide substitution than other gene duplications [137]. Y-linked duplicons make up approximately 35% of the Y chromosome; 25% of these duplicons are large inverted repeats (palindromes) [138]. Eight palindromes comprise one-quarter of the euchromatic DNA of the malespecific region of the human Y chromosome. They contain many testis-specific genes and typically exhibit 99.97% intra-palindromic (arm-to-arm) sequence identity [139]. Analysis of male-specific Y-palindrome sequence variation in existing human

Gericke populations provides evidence of recurrent arm-toarm gene conversion in our species. During recent evolution, an average of approximately 600 nucleotides per newborn male have undergone Y–Y gene conversion, which has had an important role in the evolution of multicopy testis gene families in the male-specific Y region. A high degree of Y-palindrome arm identity was interpreted as evidence that these palindromes arose through duplication events that occurred about 100,000 years ago, a feature which may link with other changes speculated in the introduction to this paper to have occurred in human neuronal network evolution at about the same time [140]. Gene conversion promoted by palindromic sequences represents a recombinatorial mechanism which transfers genetic information from a donor into a recipient gene [141]. In addition, protamines (sperm nuclear basic proteins) have simultaneously been subject to fast evolutionary positive Darwinian selection process which enhanced sperm DNA packing efficiency and chromatin stability [142].

Information load management A significant proportion of recent retrocopies in the lineage leading to humans, generated by the high rate of retroduplication in primates, seem to represent bona fide human genes. The majority are specifically expressed in testis and evolved functional roles in spermatogenesis or acquired a new or more adapted function driven by positive selection [139]. Amino acid sequence comparisons between all of 25,193 human proteins by using blast software (National Center for Biotechnology Information) demonstrate the overwhelming importance of gene regional duplication forming families of proteins with related domains and the magnitude of the set of relationships leads to the conclusion that the principal process by which new gene functions arise has been by making use of preexisting genes [143]. Could retroelements assist to limit individual gene growth through increased transcriptional networking in a process to recruit more crosstalking genes? It could be that large CFS associated human brain genes may represent more recently assembled, vulnerable evolutionary structures reflecting interaction with retroelements participating in stress responses and repair mechanisms. Does the natural history of a rearranged ‘new’ gene consist of a period of growth, followed by pruning? TEs do not make genomes grow passively as was previously thought, but seem to be controlled by forces shaping genome compactness, most likely linked to the efficiency of gene expression [144].

An integrative view of dynamic genomic elements influencing human brain evolution

RNA information caches in humans? Genomic changes in V gene structure, created by RAG recombinase acting on germline recombination signal sequences, led ultimately to immunoglobulin sequences that evolved away from immune function [51]. Only about half of the human germline V repertoire has been used in mature V(D)J rearrangement, so there appears to be further capacity for information storage. Has a critical cell cycle informational load already been responsible for the primate to human brain neoteny switch resulting in prolonged postnatal neurodevelopment? This could imply that there is a need for the germline to play a major role in informational silencing of a maintained excess informational load and that this could theoretically be stored in RNA caches and managed as RNA mediated epigenetic programming of genome rearrangement pathways, broadly similar to findings in the ciliate Oxytricha trifallax [145]. Does this imply a redundancy of available silenced epigenetic information to serve as a repository for future use? Most germline-specific genes are methylated in somatic cells [146], representing some analogy to a ‘collective unconscious’ concept. Brain specific hypomethylation and instability may allow activation of some of this information and also open the possibility of individual specific structural genetic rearrangement consequent to learning. Analysis of sequenced mutations in 1500 interspersed Alu repeats of human DNA indicates that there is a set of particular suppressed changes at certain positions that are clustered together in what appear to be sites for protein binding. The suppression of mutation appears to result from selection that is not due to requirements for Alu sequence replication. The implication is that hundreds of thousands of Alu sequences have sequence-dependent functions in the genome that are selectively important [147]. Together with other extended heterochromatic blocks in the genome, the Y might be related to the establishment of a system of gene regulation driven by natural selection [146]. The nature of imprinted genes poised between transcriptionally active and silent states, makes them good candidates for incorporation into the evolution of transgenerational adaption systems [148].

Some further considerations arising from the instability model Will the narrow margins of safety in the high risk processes utilised by RAG breakage, chromosomal rearrangement and brain ROS signaling eventually

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cause humans to become the victims of their success in a toxic environment increasingly hostile to the instability principle which has brought us thus far? Will the greatly diversified human behavioural repertoire that benefited germline diversity up to a certain stage eventually lead to such an increased level of instability related neurobehavioural disorders that it will implode our species through escalating conflict, warfare, terrorism, substance dependence problems, pollution and general destruction of the environment which sustains us? Can therapeutic manipulation of chromatin states by newer epigenetic code targeting drugs attempting to modify cell memory [149] in prereproductive individuals affect the expression of particular disorders in future generations? For instance, anti-tumour drugs have been developed that cause region-specific damage of human genomic DNA by targeting of critical repetitive sequences, such as AT-rich MAR domains [150]. Given our lack of understanding concerning the fine distinction between the physiological roles and pathological potential of CFS, much remains to be clarified during the further development of drugs targeting key evolutionary structures with possible germline consequences. Does the same hold true for the way we conduct our lives and is braingermline epigenetic instability/plasticity the basis for sporting talents or musical abilities running through a few subsequent generations in the same families? Could chaotic chromatin dynamics lead to a bifurcation point in the human germline, reproductively isolating certain current extinction risk instability carriers from those individuals carrying the fruits of the unstable period forward in a programme better able to monitor and repair the damage component of increased plasticity? Could evolutionary splitting of lineages leading to extinction of one line of previous reproductive partners have happened before in our evolutionary history, again relegating some of us in the near future to the fate of, for instance, the Neanderthals? Alternatively, does the proposed germline-brain system regulating the unstable genomic interface with the environment contain sufficient innate homeostatic ability to return to a more stable equilibrium state when a crucial instability threshold has been reached when it becomes essential for human survival? How failsafe is the methylation mechanism in itself? Another possibility is that instability has already been repeatedly tested in prehominid lineages, and an improved system of checks and balances for genomic instability has co-evolved ensuring an improved benefit:risk ratio in H sapiens. Will our escape into the future and continued intellectual development have to be based on

370 developing our own means to understand and control the soma-germline balance between plasticity/instability and/or by extending the human– machine interface? Should we rather focus solely on improving the environment as a possibly more manageable component of the brain – environment feedback loop? If genome instability over many domains of life has been stretched to the limit by the extremely genotoxic environments created by human activity, we may urgently need a better understanding of the implications of genomic instability for the continued existence of life and biodiversity generally and for our own survival as a species.

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