Epigenetics and Beyond
ISIS Report 28/01/09
Rewriting the Genetic Text in Human Brain Development
How adaptive epigenetic changes that can rewrite genes contribute to human
brain development and evolution Dr.
Mae-Wan Ho
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referenced version of this article is posted on ISIS members’ website. Details
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What makes brainy primates?
The brains of higher animals become increasingly complex in the course of
evolution, reaching a pinnacle in primates and the human species. And among
the most tantalizing discoveries since the sequencing of the human and other
genomes are the genetic and epigenetic events associated with the evolution
and development of the human brain.
Two classes of coincidental events stand out in the evolution
of primates, the end result of which is to greatly expand the diversity of
transcripts and proteins and to build the complex regulatory architecture
required for human intellectual capacity. The first is the dramatic
increase in RNA editing, a process that systematically alters the genetic
messages transcribed from the genome, creating new coding and non-coding RNAs,
and hence new proteins as well as RNAi (interfering RNA) species that regulate
networks of genes. The second is the expansion of primate-specific Alu retrotransposons,
which multiply through RNA intermediates that are reverse-transcribed and
inserted into the genome. It so happens that the increase in RNA editing in
primates occurs almost entirely within primate-specific Alu elements.
John Mattick at University of Queensland St. Lucia in Australia
and Mark Mehler at Albert Einstein College of Medicine, New York in the United
States have recently speculated that this coincidental increase in RNA editing
and Alu elements is indeed involved in the evolution and development of brainy
primates and especially of human intelligence [1].
RNA editing in evolution
The most common form of RNA editing is to change adenosine to inosine (A
to I), the I being recognized as G. This can cancel out a stop codon to create
a read through message, alter the codon, or create new splice sites in introns
(non-coding intervening sequences in interrupted genes) resulting in new exons
(coding sequences for proteins in interrupted genes). The editing is catalyzed
by members of the enzyme family adenosine deaminase that act on RNA (ADARs),
which specifically recognize double-stranded RNA longer than 30 bps (basepairs).
In reality, RNA editing encompasses a broad range of other RNA
modifications, including insertion and deletion of nucleotides that change
the entire reading frame of proteins.
RNA editing can act in concert with alternative splicing in interrupted
genes to further enhance transcript diversity [2]. For example, in the para
locus encoding a Drosophila voltage-gated Na+ channel, two
dozen processing sites for alternative splicing and RNA editing can potentially
combine to generating more than two million ‘isoforms’ of the protein.
RNA editing occurs in all taxonomic groups of organisms, but
increases dramatically in vertebrate, mammals and primates, with humans exhibiting
the highest levels of edited and multiply-edited transcripts. RNA editing
occurs in most, if not all tissues, but is particularly active in the nervous
system, where transcripts encoding proteins involved in fast neural transmission,
such as ion channels and ligand-gated receptors [1, 2]. These species-specific
alterations have profound importance for normal nervous system function.
A to I editing is much more abundant in humans than in mice,
and over 90 percent of this increased editing occurs in Alu elements
in mainly noncoding regions of RNAs, i.e., in untranslated regions (UTRs)
of mRNAs, in introns and intergenic transcripts.
ADARs have been shown to regulate neuronal gene expression through
a variety of disparate processes including modulation of RNAi, creation of
alternative splice sites, and abolition of stop codons. In addition ADARs
have a novel role in primates in the widespread editing of Alu elements.
Robert Reenan and James Jepson at Brown University Providence
Rhode Island in the United States share the view that the widespread editing
of Alu elements [2] “may well have been fundamental to the evolution of complex
behaviour.”
RNA editing in embryonic and cognitive development
RNA editing has all the appearance of being crucial in development, especially
of cognitive functions. For example, the loss of A to I editing in mice lacking
the editing enzyme ADAR1 die at the embryonic stage from defects in the production
of red blood cells and stress-induced programmed cell death, and degeneration
of the liver. Mice lacking a second editing enzyme ADAR2 exhibit profound
epileptic seizures and die shortly after birth.
In Drosophila, deletion of the single adar locus
generates morphologically wild-type adult flies that display a range of behavioural
abnormalities including severe non-coordination, temperature-sensitive paralysis,
seizures and a complete lack of courtship displays and mating. Deletion of
the RNA editing enzymes ADR1 and ADR2 in C. elegans similarly results
in chemosensory defects.
RNA editing alters transcripts from genes encoding proteins involved
in neural cell identity, maturation and function, as well as in DNA repair.
This implies that RNA editing has a role not only in neural transmission and
network plasticity but also in brain development.
In humans, three ADARs (1-3) exist, all preferentially expressed
in the nervous system, with ADAR3 being expressed exclusively in the brain.
Within the brain, ADARs exhibit complex profiles of spatiotemporal regulation
and dynamic changes in subscellular localization, and are themselves subject
to alternative splicing. Moreover, the activities of ADARs are modulated by
environmental cues, and modify signalling cues embedded within signal-transduction
pathways containing edited targets. RNA editing is not only critical for cognitive
behaviour; the deregulation of ADAR activity and associated hyper- or hypo-editing
of RNA transcripts is associated with an increased risk of neurodegenerative
disease and cancer, and neuro-developmental and psychiatric diseases in humans
[1].
Mattick and Mehler suggest that “productive” epigenetic changes
resulting from RNA editing are communicated back to the genome of neurons,
constituting the molecular basis of long-term memory and higher-order cognition.
There are at least 3 distinct ways that RNA editing can alter
brain function in response to experience (learning) and contribute to the
evolution of higher-order cognitive capacities. First by selectively editing
codons and splicing signals in protein-coding sequences involved in modulating
fast neurotransmission, the firing properties of neurons can be fine-tuned
for appropriate neuronal output and neural network integration. Second, RNA
editing can alter the processing properties and target specificities of microRNAs
(miRNAs) and the RNA interference regulatory networks in which they participate.
Third, RNA editing can modify the sequences and biophysical properties of
a vast array of other gene products, notably pre-mRNAs and the large numbers
of non-coding RNAs known to be specifically expressed in the brain and to
play roles in many functional and regulatory pathways, including epigenetic
phenomena associated with learning.
RNA editing and RNA interference
Numerous findings are suggesting that RNA editing is widespread in the brain,
affecting not only the function of individual genes in the short-term, but
its high-level long-term regulatory architecture that determines the epigenetic
states of multiple networks of genes. This occurs through interactions between
RNA editing and another ubiquitous epigenetic process, RNA interference [2].
RNA interference (RNAi) is present in all organisms that silence
genes as well as viruses and transposons in the genome [3] (see Subverting
the Genetic Text, SiS 24).. The triggers for RNAi are a range of
small RNAs ranging from 21 to 29 nt (nucleotides) in length, mainly short
interfering RNAs (siRNAs) and microRNAs (miRNAs). SiRNA and miRNA are generated
from double stranded RNA (dsRNA) precursors that are bound and cleaved by
members of the Dicer family of nucleases into small effector molecules. In
the case of siRNAs, the dsRNAs are thought to originate from inversely orientated
complementary viral or transposon sequences. SiRNA and miRNA function in similar
ways by binding to mRNA that exhibit short stretches of complementary base
sequences, resulting in cleavage of the mRNA or the inhibition of translation
into protein.
MiRNA interference has recently been shown to control a wide
variety of neurologically important processes in both vertebrates and invertebrates
[2] including neuronal expression of chemoreceptor genes, neuron-specific
splicing, circadian rhythms, morphogenesis of dendritic spine, learning and
memory. In parallel, ADAR activity may be involved in negatively regulating
the producing of miRNAs at multiple steps during maturation, and in redirectinig
the actions of miRNAs by altering their targets.
Recent research has shown that ADARs may act to inhibit the actions
of siRNAs through both their editing and dsRNA-binding activities. It is the
unrestrained actions of siRNAs and/or miRNAs that result in the chemosensory
defects of C. elegans lacking their RNA editing enzymes (see above).
Editing of miRNAs and other non-protein-coding RNAs that can
redirect the miRNAs to different targets are involved in higher order cognitive
functions dependent on entire suites of genes. The sites of RNA editing might
also be sites of small nucleolar RNA-mediated RNA modification, again suggesting
that editing is involved in genetic and epigenetic regulatory networks [1].
.
Some miRNAs subject to ADAR editing are derived from primate-specific
Alu sequences, and recent results show that miRNA-mediated translational
repression can be relieved by another class of editing enzymes, the APOBEC
family members.
Given the abundance of miRNAs in the nervous system and their
central roles in brain development, the fact that many miRNAs are derived
from introns, and that many are primate specific, RNA editing for regulatory
purposes might be widespread, and it is such high-level regulatory architecture
that controls brain development and plasticity.
Primate-specific Alu elements are the most common substrate for primate and
human ADARs
RNA A to I editing is much more abundant in humans than in mice, and over
90 percent of this increase occurs in head-to-tail repeat Alu elements in
mainly noncoding regions of RNAs, i.e., in untranslated regions of mRNAs and
in introns and intergenic transcripts.
Alu elements represent a subclass of primate-specific short interspersed
nuclear elements (SINEs) derived from 7S and tRNA sequences and spread around
the genome by retrotransposition. Three successive waves of Alu expansion
occurred during primate evolution resulting in more than 1.1 million copies
in the human genome. The major waves consist of the old AluJ subfamily about
81 million years ago, the middle-aged AluS subfamily about 48-19mya, and the
young, and still active AluY wave starting about 6 mya and continuing [4].
The possibility that Alu elements play a key role in the evolution
of human cognitive capacity via RNA A to I editing is suggested by a number
of observations [1]: RNA editing is most active in brain and important to
brain function; humans show two orders of magnitude more editing than mice;
most of the increased editing occurs in primate-specific Alu elements; and
primates have experienced the highest evolution of cognitive capacities.
Several research groups used large scale analyses to identify novel human RNA
editing sites among expressed sequences, and came up with 19 116 sites in 1
919 mRNAs, averaged over three estimates [1]. Almost all sites are within Alu
elements inserted in inverse orientations. Editing sites are not evenly distributed
along the Alu elements, but occur in hotspots, particularly the A residues at
positions 27, 29, 136 and 162. In addition, editing is tissue-specific, with
higher levels in the thymus and the brain.
Transcript of numerous genes involved in brain function subject to RNA editing
Analysis of the human RNA edited transcript databases reveal that transcripts
from gene involved in fast neural transmission represent only a small subset
of A to I RNA editing [1]. The thousands of targets include many transcripts
from genes involved in nervous system development, encompassing proteins that
modulate neural induction, and in three-dimensional patterning of the anterior
portion of the evolving neural tube and the forebrain. Also edited are transcripts
from genes involved in neural stem cell self-renewal, asymmetric cell division
and modulation of proliferation, and early neuroblast development, including
cell-cycle kinetics and migration. Other genes with edited transcripts are
involved in neural maturation including differentiation, morphogenesis, polarity,
axon guidance, dendrite formation, synapse formation, neural subtype specification
and network connectivity. Also included are transcripts from genes encoding
protocadherin a and protocadherin
b subclasses of cell-surface molecules.
Genes enoding protocadherins have been strongly implicated in the formation
of neural circuitry by encoding an unusually large repertoire of isoforms
that appear to provide the cellular address codes for directing appropriate
cell-cell interactions during progressive stages of nervous system development.
Edited transcripts include protein-coding genes that play central
roles in an extraordinary range of innovations in mature neural function:
neurone survival, excitability, signal transduction, plasticity, axodentritic
transport, energy metabolism, cell-cell and cell-environment interactions,
organization of neuronal microdomains, signalling scaffolds and cooperative
clustering of synaptic neural receptor subtypes. Many of these genes are associated
with neurodegenerative diseases and brain tumours as well as neurodevelopment
syndromes and psychiatric disorders.
These observations imply that not only synaptic strength but
also brain development is influenced by environment and experience. Moreover
if RNA editing is context dependent, this might explain the trafficking of
non-coding RNAs (ncRNAs) and mRNA to the periphery of axons and dendrites
where editing might be taking place in response to local cues, coincident
with the activation of RNA regulatory networks, and before protein translation.
RNA editing of DNA surveillance and repair enzymes and rewriting DNA
Intriguingly, transcripts from genes encoding a broad range of DNA surveillance
and repair enzymes are also subject to RNA editing [1].
Learning and memory in the brain is similar to the immune response
in many ways. A key feature of the immune system is the alteration of DNA
sequence in the genome to generate receptor diversity, in part catalyzed by
the APOBEC family of cytidine deaminases that can catalyze cytosine to uracil
(C to U) and cytosine to thymine (C to T) editing of RNA and DNA.
The possibility exists that DNA recoding – rewriting genome DNA
- is a central feature of both the immune and nervous systems. DNA recoding
may be involved at the level of establishing neuronal identity and neuronal
connectivity during development, learning and brain regeneration. And it appears
that the brain, like the immune system, also changes according to experience.
Mattick and Mehler suggest that the potential recoding of DNA
in nerve cells (and similarly in immune cells) might be primarily a mechanism
whereby productive or learned changes induced by RNA editing are rewritten
back to DNA via RNA-directed DNA repair. (See the latest model of RNA-directed
recoding of DNA proposed for the immune system [5] by Ted Steele at Australian
National University Canberra). This effectively fixes the altered genetic
message once a particular neural circuitry and epigenetic state has been established.
The suggestion that memory formation involves RNA-directed DNA
modifications similar to those in the immune system is supported by a range
of circumstantial observations over many years. For example, two enzymes involved
in generating diversity in the immune system (Rag1 and Rag2) are expressed
in the central nervous system and in olfactory sensory neurons that are actively
involved in experience-mediated neural plasticity. Furthermore, recombination
catalyzed by Rag1 and Rag2 and programmed genomic rearrangements in other
organisms are RNA directed, although it remains uncertain whether such recombination
occurs in the brain and is relevant to brain function.
Members of the DNA polymerase Y family involved in somatic hypermutation
of genes encoding immunoglobulins have reverse transcriptase activity [6].
One of them, DNA polymerase-i is expressed in areas of the brain associated
with learning and memory, as is DNA polymerase M, which is involved in rearrangement
of genes encoding immunoglulins. The fact that transcripts from genes encoding
enzymes putatively involved in DNA recoding are themselves edited suggests
that the process is subject to contextual control, which might explain why
some memories are more vivid and enduring than others. Moreover, A to G mutations
correlate with nascent mRNA hairpin at somatic hypermutation hotspots, implying
roles for both RNA editing and reverse transcription during somatic hypermutation
[7], involving mismatch repair enzymes that are expressed in the hippocampus.
It has been shown recently that RNA-directed DNA repair can occur
in eukaryotic cells. In addition, LINE1 elements that are active in the human
genome encode several proteins, including a reverse transcriptase, and individual
SINE elements including active Alu sequences can hijack and use the LINE1
reverse transcriptase.
The suggestion that there might be communication of RNA-encoded
information back to the genome at the epigenetic and genetic levels would
also potentially explain the surprising observation that diverse RNA species
and associated regulatory signals are not only trafficked to the periphery
of the nerve cell, but might also undergo retro-transport back to the nucleus.
There is increasing evidence for retrograde transport of RNAs, including small
RNAs, to the nucleus in a broad range of organisms, as well as for RNA informational
exchange between cells through ‘exosomes’, specific RNA receptors and derivation
of presynaptic RNA from surrounding glial cells.
There are clear evolutionary and functional parallels between
members of the immunoglobulin (Ig) superfamily and the protocadherins, as
well as many other subclasses of nervous system-selective Ig superfamily domain-containing
proteins involved in neuronal cell identity, connectivity, synaptic plasticity
and developmental and adult brain homeostasis.
The pervasive presence of a broad array of functional subclasses
of Ig-like CNS superfamily proteins might represent flexible modules for molecular
recognition and also particularly amenable targets of APOBEC-mediated editing/mutation,
as they are in the immune system.
APOBEC enzymes, as well as ADARs, also exhibit dynamic changes
in nuclear-cytoplasmic translocation, intranuclear microdomain localization
and editing functions in response to the environment. The APOBEC3 subfamily
has been vastly expanded in primates, and complexes formed with APOBEC3 are
recruited into RNA transport granules that contain both Staufen (RNA-binding
protein) and Alu sequences. Staufen has been shown to be required for long-term
memory formation in Drosophila, as has Armitage, a putative RNA helicase
required for mRNA transport and translation at the synapse.
Numerous observations bear out the pervasive role of RNA editing
and its potential coupling to DNA recoding via RNA trafficking between nucleus
and synapse, and RNA-templated DNA repair enzymes in the evolution, development
and function of the human brain.
Mattick and Mehler suggest that environmentally induced changes
in neural development and brain architecture, cell identity and synaptic connectivity
might become “hardwired in the genome”, “potentially defining the complex
and emergent properties of long-term memories and other structural and functional
adaptations.
“If correct, this hypothesis predicts that individual neural
cells will, in fact, have distinctive spatially and temporally defined genomic
sequences and chromatin structure… It also predicts that memory consolidation,
storage and retrieval and associated long-term adaptations of human brain
form and function should be modifiable by the targeted and differential modulation
of expression of genes encoding enzymes involved in RNA editing and DNA recoding.”
Sperm-mediated gene transfer, the inheritance of acquired characters
Mattick and Mehler fall short of proposing that the RNA-templated recoding
of the genome and the associated structural and functional adaptations could
be transmitted to the next generation. This would appear to be crucial for
brain evolution in primates leading up to humans, so that the gains made by
each generation could be accumulated.
If the analogy with the immune system holds, then as suggested by Steele and
colleagues, edited RNA messages or their reverse transcribed DNA counterparts
could become inherited via the sperm [5, 7, 8] (see Epigenetic
Inheritance Through Sperm Cells, SiS 41).
“Sperm-mediated gene transfer” has been well documented by Italian
researcher Corrado Spadafora [9] as a process whereby new genetic traits are
transmitted to the next generation by the uptake of DNA or RNA by spermatozoa
and delivered to the oocytes at fertilization. The interaction of exogenous
nucleic acids with sperm cells is mediated by specific factors, among which,
a reverse transcriptase that generates “retro-genes” through reverse transcription
of exogenous RNA or through sequential transcription, splicing and reverse
transcription of exogenous DNA. The result is to transmit low copy transcriptionally
active extrachromosomal structures capable of determining new traits. Retro-genes
can be further transmitted through sexual reproduction from founders to their
F1 progeny as new genetic and phenotypic features, unlinked to chromosomes,
and thus be generated and inherited in a non-Mendelian manner. Rare instances
of retro-gene integration into the chromosome could also occur, providing
further potential for evolution.
I thank Ted Steele for comments on earlier drafts of this article.
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