**4.1 How it all comes together: A flexible networking "interface system"**

The ability to alter development, physiology, growth, and behaviour in response to different environmental conditions represent critical assessments of both external and internal factors as a function of energy balance and environmental stress as well as physiological, developmental, and behavioural responses to these determinations (Crespi & Denver, 2005). What may have made humans unique, is an enormously increased feedback capability for constructive interaction between internal structures and extra-biological factors, self reflective evaluation and an improved ability to shape the environment, to such an extent that there is now an unprecedented information continuum between information captured in biological processes and the environment itself. In support of more holistic modelling, integrative biological concepts have also arisen, for instance the concept of a "neuroimmuno-endocrine system". It is additionally proposed here that the flexible disparate stress response mechanisms responsible for the storage of novel information should be considered as a combinatorially regulated "interface system". It can be expected that an evolutionary strategy would exploit the effect of integrating the different systems for the transmission of genetic information with systems relating to the external adaptation of the organism (Bengtsson, 2004). An interface can be defined as a point at which independent systems or diverse groups interact. Although such a proposed environmental interface system presumably consists of many more components than mentioned here, an example will be provided of a nonlinear brain system with flexible internal structural responses to environmentally induced perturbation i.e. the brain chromosomal 'fragilome' which appears to underly certain aspects of neuroplasticity and memory storage and which appears to be closely linked with the stress hormonal system and immunoglobulin DNA strand break and genomic rearranging phenomena. The essential important characteristic of the brain fragilome is that of genetic breakage and recombination offering a structural basis for genetic/neuronal diversification and storage of memories (Gericke, 2010).

Chromosomal fragile sites represent large heritable chromosomal regions that preferentially exhibit gaps or breaks after DNA synthesis is partially perturbed by stressors affecting the replication process (Arlt et al., 2006) and are classified as 'rare' or 'common', depending on their induction method and frequency within the population. Common fragile sites (CFS) are found in all individuals, are variable, extend over large regions and are associated with transcriptional activity (Sbrana et al., 1998).

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. Chromosomal fragile sites (*in toto* represented by the fragilome), represent *in vitro* observed genomic regions with particular structural characteristics related to epigenetic plasticity, resulting in the creation of diversity through a process of controlled double strand breaks and imperfect mismatch repair shielded from and/or below an apoptotic risk threshold under physiological circumstances. This modular

(Lubin & Sweatt, 2007), and RNA-mediated chromatin-level silencing is increasingly implicated in development, stress responses, and natural epigenetic variation that may promote phenotypic diversity, physiological plasticity, and evolutionary change (Madlung & Comai, 2004). Epigenetic markers on the promoter regions of the Bdnf gene have been the most extensively studied, including alterations in histone acetylation, phosphorylation, methylation, and DNA methylation associated with memory behaviour (Gupta et al., 2010), as

The ability to alter development, physiology, growth, and behaviour in response to different environmental conditions represent critical assessments of both external and internal factors as a function of energy balance and environmental stress as well as physiological, developmental, and behavioural responses to these determinations (Crespi & Denver, 2005). What may have made humans unique, is an enormously increased feedback capability for constructive interaction between internal structures and extra-biological factors, self reflective evaluation and an improved ability to shape the environment, to such an extent that there is now an unprecedented information continuum between information captured in biological processes and the environment itself. In support of more holistic modelling, integrative biological concepts have also arisen, for instance the concept of a "neuroimmuno-endocrine system". It is additionally proposed here that the flexible disparate stress response mechanisms responsible for the storage of novel information should be considered as a combinatorially regulated "interface system". It can be expected that an evolutionary strategy would exploit the effect of integrating the different systems for the transmission of genetic information with systems relating to the external adaptation of the organism (Bengtsson, 2004). An interface can be defined as a point at which independent systems or diverse groups interact. Although such a proposed environmental interface system presumably consists of many more components than mentioned here, an example will be provided of a nonlinear brain system with flexible internal structural responses to environmentally induced perturbation i.e. the brain chromosomal 'fragilome' which appears to underly certain aspects of neuroplasticity and memory storage and which appears to be closely linked with the stress hormonal system and immunoglobulin DNA strand break and genomic rearranging phenomena. The essential important characteristic of the brain fragilome is that of genetic breakage and recombination offering a structural basis for

well as activity-dependent changes in DNA methylation (Nelson et al., 2008).

**4.1 How it all comes together: A flexible networking "interface system"** 

genetic/neuronal diversification and storage of memories (Gericke, 2010).

transcriptional activity (Sbrana et al., 1998).

Chromosomal fragile sites represent large heritable chromosomal regions that preferentially exhibit gaps or breaks after DNA synthesis is partially perturbed by stressors affecting the replication process (Arlt et al., 2006) and are classified as 'rare' or 'common', depending on their induction method and frequency within the population. Common fragile sites (CFS) are found in all individuals, are variable, extend over large regions and are associated with

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. Chromosomal fragile sites (*in toto* represented by the fragilome), represent *in vitro* observed genomic regions with particular structural characteristics related to epigenetic plasticity, resulting in the creation of diversity through a process of controlled double strand breaks and imperfect mismatch repair shielded from and/or below an apoptotic risk threshold under physiological circumstances. This modular assembly process has been adapted from the immune paradigm at the expense of a risk for instability and/or malignant transformation when associated control mechanisms are not in place (such as tumour suppressor genes). It is suggested that the abilities for diverse recognition of externally derived information, a dynamic response of somatic hypermutation followed by genome rearrangement creating a template for memory formation, and entry into a terminally differentiated state are features common to both the brain and immune system. Chromosome breaks and the various resulting structural rearrangements (genetic instability) have mostly been viewed in a pathological context by researchers, but controlled chromosomal breakage and rearrangement leading to altered gene expression without adverse effects may have been necessary for the evolutionary and neurodevelopmental flexibility required by the human brain (Gericke, 2010).

Such chromosomal breakage relates to alterations in DNA higher order structures and studies of fragile sites at the level of chromosome organization reveal an unusual chromatin structure associated with fragile sites influencing formation of nucleosomes and the formation of nucleosome arrays (Wang, 2006). The study of epigenetics focuses on the relationship between chromatin structure and gene transcription. DNA is commonly packaged into nucleosomes and wrapped tightly around a core of histone (H) proteins. Modifications that regulate chromatin structure influence transcriptional activity, in part, through effects on transcription factor binding. the environmental regulation of histone methylation states. Chromatin remodeling and gene transcription are linked in that transcriptional activation associates with chromatin states that enhance the probability of subsequent transcriptional activity, providing a feed-forward loop. (Cordero et al., 2006, Murr, 2010; Hayashi et al., 2011). Recent observations reveal that histones are removed and replaced to enable or restrict, respectively, access of the transcription machinery to regulate transcription. The ultimate goal of some epigenetic modifications might well turn out to be the regulation of histone occupancy on the DNA (Williams et al., 2008). CFS-associated duplication and deletion altering AT tract length and DNA flexibility have been linked with variation in nucleosomal architecture (Cosgrove & Walberger, 2005). AT-rich repeats mediate recombination events in non-homologous chromosomes during meiosis (Jackson et al., 2003) and due to a modification of binding factor characteristics, CFS have been proposed to contribute to epigenetic sensitive phenotypes (Woynarowski, 2004), a phenomenon which has been suggested to include neurobehavioral effects (Garofalo et al.,1993; Gericke et al., 1995, Gericke 1998, 2006, 2010; Simonic & Gericke 1996; Simonic & Ott, 1996; Savelyeva et al., 2006).

#### **4.2 Developmental cytogenetic instability in the mammalian brain**

"As many of the examples of epigenetic inheritance are mediated by position effects, the possibility exists that chromosome rearrangements may be one of the driving forces behind evolutionary change by exerting position effect alterations in gene activity, an idea first articulated by Richard Goldschmidt in 1940 in his book "The Material Basis of Evolution"(Reprinted in 1982).

The emerging evidence suggests that Goldschmidt's controversial hypothesis deserves a serious reevaluation" (Varmuza, 2003). Recent findings of rearranged and aneuploid chromosomes in brain cells suggest an unexpected link between developmental chromosomal instability and brain genome diversity (Yurov et al., 2007), (Yang et al., 2003). In humans, previously unrecognized large-scale double-stranded DNA breaks are now known to occur under normal circumstances in early postmitotic and differentiating

Stress Shaping Brains: Higher Order DNA/Chromosome

particular individual.

brain.

Mechanisms Underlying Epigenetic Programming of the Brain Transcriptome 357

expressing a different protocadherin. In this way, 4,950 different neurons arising from one stem cell form a neuronal network in which homophilic contacts can be formed in 52 layers, permitting an enormous number of different connections between neurons (Wu, 2005). At the single-cell level, protocadherin-alpha mRNAs are regulated monoallelically, supporting the idea that diversified protocadherin molecules contribute to neural circuit development and provide individual cells with their specific identity (Hilschmann et al., 2001). The neocortical genomic response to stress is relayed via hormones and reactive oxygen/nitrogen species signaling, thereby implicating the mitochondrial genome and bioenergetic metabolism (Wallace, 2010), which is suggested to represent an extension of dynamic genomic changes in parallel to the immune recombination and neural rearrangement (protocadherin) histories and fragile site events (Gericke, 2006) of a

**5. Stress memory and immune-like rearrangement in the human brain** 

Doubts have more recently been raised whether gene transcription activated by dendritic calcium signals is sufficient to consolidate long-term functional alterations associated with memory consolidation. An alternative genomic hypothesis of memory suggests that acquired information is persistently stored within individual neurons through modifications of DNA, and that these modifications serve as the carriers of elementary memory traces. The emerging idea is therefore that lifelong behavioural memory storage may involve lasting changes in the physical, three-dimensional structure of DNA itself and chromatin alterations are emerging as a key epigenetic mechanism in the process in conjunction with usedependent synaptic plasticity (Levenson & Sweatt, 2006; Delcuve et al., 2009). The expression of immune recombination activating genes in key stress-induced memory regions in the brain suggests the adoption by the brain of this ancient pattern recognition and memory system to establish a structural basis for long-term memory through controlled chromosomal breakage at highly specific genomic regions. Fundamentally unstable processes with narrow safety margins (controlled chromosomal breakage) thus appear to underlie pattern recognition and memory consolidation in both the immune system and

Unusual genetic mechanisms for diversifying recognition proteins may be a widespread characteristic of animal immunity and may have paved the way for adaptation for management of neural sensory information (Litman et al., 2005). Stress reactions form part of neuroendocrine influences that also modulate immune function. The appearance of a lymphocyte-based recombinatorial system of anticipatory immunity in vertebrates approximately 500 mya facilitated developmental and morphological plasticity in addition to the advantage conferred by the ability to recognize a larger portion of the antigenic world (Pancer & Cooper, 2006). A prototypic example of epigenetic-facilitation in memory retention pertains to memory T-cells of the mammalian immune system (reviewed in Nakayama & Yamashita (2008)). Numerous epigenetic mechanisms such as histone modifications and DNA methylation modulate gene expression and thus play a role in T-cell survival and maintenance of T-cell function in various differentiated states. These processes underlie the formation of persistent immunological memory cells in response to transient environmental stimuli (reviewed in Nakayama & Yamashita (2008). Thus, like immune Tcells, it is plausible that epigenetic mechanisms such as methylation of the cytosine base are changeable and occur in post-mitotic neurons to mediate neuronal function. However,

neurons (Gilmore et al., 2000). In general, accumulation of DNA breaks in differentiating cells cannot be attributed to a decrease in the DNA repair efficiency. Poly(ADP)ribose synthesis often follows the DNA breakage in differentiating cells. It has been hypothesized that DNA fragmentation is an epigenetic tool for regulating the differentiation process (Sjakste & Sjakste, 2007). Genomes of developing and adult neurons can be different at the level of whole chromosomes (Rehen et al., 2005). 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, similar to processes involved in genetic diversification in plants. 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 in the adult mammalian brain, investigations suggest that the loss or gain of chromosomes might provide a mechanism to regulate gene expression during development of new cells in the adult vertebrate brain (Rajendran et al., 2007),(Yurov et al., 2005). Both neurons and non-neuronal cells can be aneuploid as a normal feature of the human brain (Rehen et al., 2005).

One possible consequence of nervous system aneuploidy is altered gene expression through loss of heterozygosity (Kaushal et al., 2003). Aneuploid neurons were found to be functionally active and demonstrate that functioning neurons with aneuploid genomes form genetically mosaic neural circuitries as part of the normal organization of the mammalian brain (Kingsbury et al., 2005). 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*  (Yurov et al., 2007) 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. This appears to be different from altered chromosomal breakage which can be demonstrated in peripheral blood and may reflect more widespread gene expression changes.

#### **4.3 Protocadherin genetic rearrangement in the brain**

Both the immune system and the brain evolved from a cell adhesion system. 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. Cadherin-related neuronal receptor/protocadherin transcript variance has also been linked with chromosomal variations in the nucleus of differentiated neurons (Yagi, 2003). Together with cytoskeletal proteins, such as tubulin, microtubule-associated proteins, and intermediate filament proteins, the neural adhesive protocadherins with immunoglobulin-like functional features and extracellular matrix glycoproteins are associated with dynamic structural remodelling in the nervous system (Miyate & Hatton, 2002; Chun 1999). Some brain protocadherins are specific to the hominoid lineage (Durand et al, 2006) and single nucleotide polymorphisms in the protocadherinalpha and –beta genes are possible contributors to variation in human brain function (Pedrosa et al., 2008). Furthermore, different codons in the mammalian protocadherin ectodomains are under diversifying selection. These diversified residues likely play an important role in combinatorial interactions, which could provide the staggering diversity required for neuronal connections in the brain (Miki et al., 2005).

While lymphocytes express a single receptor molecule specifically directed against an outside stimulus, in contrast, each neuron has three specific recognition sites, each

neurons (Gilmore et al., 2000). In general, accumulation of DNA breaks in differentiating cells cannot be attributed to a decrease in the DNA repair efficiency. Poly(ADP)ribose synthesis often follows the DNA breakage in differentiating cells. It has been hypothesized that DNA fragmentation is an epigenetic tool for regulating the differentiation process (Sjakste & Sjakste, 2007). Genomes of developing and adult neurons can be different at the level of whole chromosomes (Rehen et al., 2005). 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, similar to processes involved in genetic diversification in plants. 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 in the adult mammalian brain, investigations suggest that the loss or gain of chromosomes might provide a mechanism to regulate gene expression during development of new cells in the adult vertebrate brain (Rajendran et al., 2007),(Yurov et al., 2005). Both neurons and non-neuronal cells can be

One possible consequence of nervous system aneuploidy is altered gene expression through loss of heterozygosity (Kaushal et al., 2003). Aneuploid neurons were found to be functionally active and demonstrate that functioning neurons with aneuploid genomes form genetically mosaic neural circuitries as part of the normal organization of the mammalian brain (Kingsbury et al., 2005). 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*  (Yurov et al., 2007) 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. This appears to be different from altered chromosomal breakage which can be demonstrated in peripheral blood and may

Both the immune system and the brain evolved from a cell adhesion system. 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. Cadherin-related neuronal receptor/protocadherin transcript variance has also been linked with chromosomal variations in the nucleus of differentiated neurons (Yagi, 2003). Together with cytoskeletal proteins, such as tubulin, microtubule-associated proteins, and intermediate filament proteins, the neural adhesive protocadherins with immunoglobulin-like functional features and extracellular matrix glycoproteins are associated with dynamic structural remodelling in the nervous system (Miyate & Hatton, 2002; Chun 1999). Some brain protocadherins are specific to the hominoid lineage (Durand et al, 2006) and single nucleotide polymorphisms in the protocadherinalpha and –beta genes are possible contributors to variation in human brain function (Pedrosa et al., 2008). Furthermore, different codons in the mammalian protocadherin ectodomains are under diversifying selection. These diversified residues likely play an important role in combinatorial interactions, which could provide the staggering diversity

While lymphocytes express a single receptor molecule specifically directed against an outside stimulus, in contrast, each neuron has three specific recognition sites, each

aneuploid as a normal feature of the human brain (Rehen et al., 2005).

reflect more widespread gene expression changes.

**4.3 Protocadherin genetic rearrangement in the brain** 

required for neuronal connections in the brain (Miki et al., 2005).

expressing a different protocadherin. In this way, 4,950 different neurons arising from one stem cell form a neuronal network in which homophilic contacts can be formed in 52 layers, permitting an enormous number of different connections between neurons (Wu, 2005). At the single-cell level, protocadherin-alpha mRNAs are regulated monoallelically, supporting the idea that diversified protocadherin molecules contribute to neural circuit development and provide individual cells with their specific identity (Hilschmann et al., 2001). The neocortical genomic response to stress is relayed via hormones and reactive oxygen/nitrogen species signaling, thereby implicating the mitochondrial genome and bioenergetic metabolism (Wallace, 2010), which is suggested to represent an extension of dynamic genomic changes in parallel to the immune recombination and neural rearrangement (protocadherin) histories and fragile site events (Gericke, 2006) of a particular individual.
