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Brain cell somatic gene recombination and its phylogenetic foundations

2020; Elsevier BV; Volume: 295; Issue: 36 Linguagem: Inglês

10.1074/jbc.rev120.009192

1083-351X

Gwendolyn E. Kaeser, Jerold Chun,

RNA and protein synthesis mechanisms

A new form of somatic gene recombination (SGR) has been identified in the human brain that affects the Alzheimer's disease gene, amyloid precursor protein (APP). SGR occurs when a gene sequence is cut and recombined within a single cell's genomic DNA, generally independent of DNA replication and the cell cycle. The newly identified brain SGR produces genomic complementary DNAs (gencDNAs) lacking introns, which integrate into locations distinct from germline loci. This brief review will present an overview of likely related recombination mechanisms and genomic cDNA-like sequences that implicate evolutionary origins for brain SGR. Similarities and differences exist between brain SGR and VDJ recombination in the immune system, the first identified SGR form that now has a well-defined enzymatic machinery. Both require gene transcription, but brain SGR uses an RNA intermediate and reverse transcriptase (RT) activity, which are characteristics shared with endogenous retrotransposons. The identified gencDNAs have similarities to other cDNA-like sequences existing throughout phylogeny, including intron-less genes and inactive germline processed pseudogenes, with likely overlapping biosynthetic processes. gencDNAs arise somatically in an individual to produce multiple copies; can be functional; appear most frequently within postmitotic cells; have diverse sequences; change with age; and can change with disease state. Normally occurring brain SGR may represent a mechanism for gene optimization and long-term cellular memory, whereas its dysregulation could underlie multiple brain disorders and, potentially, other diseases like cancer. The involvement of RT activity implicates already Food and Drug Administration–approved RT inhibitors as possible near-term interventions for managing SGR-associated diseases and suggest next-generation therapeutics targeting SGR elements. A new form of somatic gene recombination (SGR) has been identified in the human brain that affects the Alzheimer's disease gene, amyloid precursor protein (APP). SGR occurs when a gene sequence is cut and recombined within a single cell's genomic DNA, generally independent of DNA replication and the cell cycle. The newly identified brain SGR produces genomic complementary DNAs (gencDNAs) lacking introns, which integrate into locations distinct from germline loci. This brief review will present an overview of likely related recombination mechanisms and genomic cDNA-like sequences that implicate evolutionary origins for brain SGR. Similarities and differences exist between brain SGR and VDJ recombination in the immune system, the first identified SGR form that now has a well-defined enzymatic machinery. Both require gene transcription, but brain SGR uses an RNA intermediate and reverse transcriptase (RT) activity, which are characteristics shared with endogenous retrotransposons. The identified gencDNAs have similarities to other cDNA-like sequences existing throughout phylogeny, including intron-less genes and inactive germline processed pseudogenes, with likely overlapping biosynthetic processes. gencDNAs arise somatically in an individual to produce multiple copies; can be functional; appear most frequently within postmitotic cells; have diverse sequences; change with age; and can change with disease state. Normally occurring brain SGR may represent a mechanism for gene optimization and long-term cellular memory, whereas its dysregulation could underlie multiple brain disorders and, potentially, other diseases like cancer. The involvement of RT activity implicates already Food and Drug Administration–approved RT inhibitors as possible near-term interventions for managing SGR-associated diseases and suggest next-generation therapeutics targeting SGR elements. 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These and most other assessments have linked large structural changes to functional consequences, albeit in the absence of precisely identified and affected cellular genes. The study of the functional consequences of somatic genomic mosaicism in the human brain has been especially difficult, hampered by both technical and biological challenges that include limitations of working with postmitotic cells and the human brain. Indeed, even identification of somatic genomic mosaicism in human brain is challenging. Issues range from access to consistent, high-quality human autopsy materials of comparable age, sex, and race or sequencing technologies and bioinformatics not optimized for mosaicism to biological impediments like nonclonal genomic alterations affecting just a single postmitotic cell, obfuscated by a sea of normal cells. For example, the brain shows functional uniqueness of very small populations of cells, such as neurons responsive to input from a particular point in visual space (30Hecker A. Schulze W. Oster J. Richter D.O. Schuster S. Removing a single neuron in a vertebrate brain forever abolishes an essential behavior.Proc. Natl. Acad. Sci. U.S.A. 2020; 117 (32001507): 3254-326010.1073/pnas.1918578117Crossref PubMed Scopus (2) Google Scholar, 31Okuyama T. Social memory engram in the hippocampus.Neurosci. Res. 2018; 129 (28577978): 17-2310.1016/j.neures.2017.05.007Crossref PubMed Scopus (15) Google Scholar) or even the proposed single "grandmother" cell that responds to a complex assembly of visual features such as those comprising a specific face (32Gross C.G. 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Moreover, each neuron has an anatomically unique location tied to function; without additional preparative and analytical steps (that do not readily exist for human brain studies), sequencing a desired single neuron would be broadly analogous to trying to isolate and sequence a desired single colon cancer stem cell by sequencing a large pool derived from all cells of the body: it could completely miss the targeted tissue let alone the single cell. Combined, these extreme examples illustrate how traditional approaches designed to assess germline changes in bulk peripheral tissues, or even focused approaches that might detect mutations at a 0.1% level, still remain many orders of magnitude away from detecting genomic changes within single targeted neurons of the human brain and highlight the importance of good study design and technology considerations for both cell isolation and reliable sequencing. The existence of diverse forms of somatic genomic mosaicism increased the possibility that brain SGR might exist, albeit in the absence of a specific gene candidate. However, a promising gene candidate emerged during more recent studies of genomic mosaicism in Alzheimer's disease (AD) brains: the amyloid precursor protein (APP) gene. This prominent AD gene has a well-known gene dosage effect, in which its increased copy number is causal for AD in rare familial AD cases and Down syndrome (trisomy 21, with three copies of APP via its location on chromosome 21). Intriguingly, APP copy numbers were found to be mosaically increased in sporadic AD neuronal genomes, with evidence for amplification in discrete locations within a nucleus (22Bushman D.M. Kaeser G.E. Siddoway B. Westra J.W. Rivera R.R. Rehen S.K. Yung Y.C. Chun J. Genomic mosaicism with increased amyloid precursor protein (APP) gene copy number in single neurons from sporadic Alzheimer's disease brains.eLife. 2015; 4 (25650802): e0511610.7554/eLife.05116Crossref Scopus (27) Google Scholar, 34Lee M.H. Siddoway B. Kaeser G.E. Segota I. Rivera R. Romanow W.J. Liu C.S. Park C. Kennedy G. Long T. Chun J. Somatic APP gene recombination in Alzheimer's disease and normal neurons.Nature. 2018; 563 (30464338): 639-64510.1038/s41586-018-0718-6Crossref PubMed Scopus (82) Google Scholar). Analyses were thus focused on APP genomic sequences in human neurons: remarkably, the identified APP sequences resembled complementary DNAs (cDNAs): they lacked introns yet were present in genomic DNA and were therefore termed "gencDNAs" (Fig. 1). Their many forms and copies represented novel components of the somatic human genome yet displayed features that were highly reminiscent of known, genetic (for single-celled organisms), or germline evolutionary elements lacking introns: intron-less genes and inactive processed pseudogenes. This review will compare and contrast gencDNAs produced by brain SGR to other phylogenic genomic elements showing gene intron loss and cDNA-like sequences. It will also broadly compare and contrast the two known types of SGR: the newly discovered brain SGR that results in gencDNA products and VDJ recombination in the immune system (in-depth reviews of VDJ recombination have been published elsewhere (35Roth D.B. V(D)J recombination: mechanism, errors, and fidelity.Microbiol. Spectr. 2014; 2 (26104458)10.1128/microbiolspec.MDNA3-0041-2014Crossref PubMed Google Scholar, 36Schatz D.G. Swanson P.C. V(D)J recombination: mechanisms of initiation.Annu. Rev. Genet. 2011; 45 (21854230): 167-20210.1146/annurev-genet-110410-132552Crossref PubMed Scopus (287) Google Scholar)). Possible mechanisms producing brain SGR along with potential roles in aging and disease are also discussed. Other forms of DNA recombination, such as DNA repair mechanisms and meiotic recombination, are beyond the scope of this review. SGR of APP manifested as many genomic APP variants present within a single human brain (34Lee M.H. Siddoway B. Kaeser G.E. Segota I. Rivera R. Romanow W.J. Liu C.S. Park C. Kennedy G. Long T. Chun J. Somatic APP gene recombination in Alzheimer's disease and normal neurons.Nature. 2018; 563 (30464338): 639-64510.1038/s41586-018-0718-6Crossref PubMed Scopus (82) Google Scholar), forming a genomic mosaic of distinct sequences and copy numbers that varied among brain cell genomes. APP gencDNA sequences lacked introns, showed the basic structure of a cDNA, and included known, brain-specific RNA splice variants, APP-751 and APP-695 (Fig. 1). The complete APP-770 form (that contains all exons) is generally not expressed in the brain and was not identified as a neuronal gencDNA. The presence of brain-specific RNA splice variant sequences as gencDNAs supports a requirement for an expressed RNA intermediate template in gencDNA formation, as well as reverse transcription of the RNA molecule. Although full-length forms were observed and enriched in nondiseased brains, most gencDNAs that were prevalent in AD brains showed truncated APP sequences containing intraexonic junctions (IEJs), in which noncontiguous exons are fused by intraexonic short microhomology sequences, ranging from 2 to 20 base pairs. When sequenced as single, intact molecules using long-read Pacific Biosciences single-molecule real-time circular consensus sequencing, gencDNA sequence diversity included not only IEJs but numerous small insertions, deletions, and, most commonly, SNVs. A marked shift in AD versus nondiseased gencDNA forms and representation was observed, involving an AD-related 10-fold increase in gencDNA diversity showing a preponderance of IEJ-containing truncated gencDNAS with many insertions, deletions, and SNVs, which contrasted with a majority of full-length sequences in nondiseased neurons. Interestingly, sporadic AD neurons contained gencDNAs with 11 known familial AD-causing SNVs that nonetheless occurred somatically. The role for gencDNAs in disease was further supported by DNA in situ hybridization that revealed from 0 to 13 gencDNA loci within a single neuronal nucleus and a much higher prevalence in diseased tissue. Notably, 30–40% of AD and 75% of nondiseased neuronal nuclei showed no evidence of APP gencDNAs, emphasizing the importance of accounting for genomic mosaicism in experimental procedures on human brain. The existence of APP gencDNAs has been independently confirmed (37Park J.S. Lee J. Jung E.S. Kim M.H. Kim I.B. Son H. Kim S. Kim S. Park Y.M. Mook-Jung I. Yu S.J. Lee J.H. Brain somatic mutations observed in Alzheimer's disease associated with aging and dysregulation of Tau phosphorylation.Nat. Commun. 2019; 10 (31300647): 309010.1038/s41467-019-11000-7Crossref PubMed Scopus (0) Google Scholar), and reanalysis of published data identified gencDNA insertion sites distributed throughout the genome and away from germline APP on chromosome 21 (38Lee M.-H. Liu C.S. Zhu Y. Kaeser G.E. Rivera R. Romanow W.J. Kihara Y. Chun J. Reply: Evidence that APP gene copy number changes reflect recombinant vector contamination.bioRxiv. 2020; 10.1101/730291Google Scholar). These findings are consistent with 1) DNA in situ hybridization signals that showed gencDNAs in nuclear locations that were distinct from the wildtype (WT) locus (34Lee M.H. Siddoway B. Kaeser G.E. Segota I. Rivera R. Romanow W.J. Liu C.S. Park C. Kennedy G. Long T. Chun J. Somatic APP gene recombination in Alzheimer's disease and normal neurons.Nature. 2018; 563 (30464338): 639-64510.1038/s41586-018-0718-6Crossref PubMed Scopus (82) Google Scholar) and 2) the previously reported increased DNA content variation observed in human cortical neurons, that is further increased in AD (22Bushman D.M. Kaeser G.E. Siddoway B. Westra J.W. Rivera R.R. Rehen S.K. Yung Y.C. Chun J. Genomic mosaicism with increased amyloid precursor protein (APP) gene copy number in single neurons from sporadic Alzheimer's disease brains.eLife. 2015; 4 (25650802): e0511610.7554/eLife.05116Crossref Scopus (27) Google Scholar, 23Westra J.W. Rivera R.R. Bushman D.M. Yung Y.C. Peterson S.E. Barral S. Chun J. Neuronal DNA content variation (DCV) with regional and individual differences in the human brain.J. Comp. Neurol. 2010; 518 (20737596): 3981-400010.1002/cne.22436Crossref PubMed Scopus (71) Google Scholar, 24Fischer H.G. Morawski M. Brückner M.K. Mittag A. Tarnok A. Arendt T. Changes in neuronal DNA content variation in the human brain during aging.Aging Cell. 2012; 11 (22510449): 628-63310.1111/j.1474-9726.2012.00826.xCrossref PubMed Scopus (46) Google Scholar) through the formation of new DNA sequences that are distinct from WT loci and germline DNA. Brain SGR undoubtedly affects other genes, and indeed, IEJs in other genes were found in an independent, commercially produced, and publicly available long-read whole-exome RNA-sequencing data set (38Lee M.-H. Liu C.S. Zhu Y. Kaeser G.E. Rivera R. Romanow W.J. Kihara Y. Chun J. Reply: Evidence that APP gene copy number changes reflect recombinant vector contamination.bioRxiv. 2020; 10.1101/730291Google Scholar, 39.(2016) Data Release: Alzheimer Brain Isoform Sequencing (Iso-Seq) Dataset.Google Scholar). However, SGR does appear to be specific to particular genes at least in AD neurons from the prefrontal cortex: notably, PSEN1, another causal AD gene in rare families, was not similarly affected by SGR (34Lee M.H. Siddoway B. Kaeser G.E. Segota I. Rivera R. Romanow W.J. Liu C.S. Park C. Kennedy G. Long T. Chun J. Somatic APP gene recombination in Alzheimer's disease and normal neurons.Nature. 2018; 563 (30464338): 639-64510.1038/s41586-018-0718-6Crossref PubMed Scopus (82) Google Scholar). The proof of concept for biological repercussions of gencDNAs already has enormous support through the widespread generation and use of exogenous cDNA transgenes, in cells and animals, which are similar in structure to gencDNAs. These are most often driven by non-WT promoters, which are routinely used for bacterial or yeast transformation, mammalian cell line transfection, retrovirally mediated cDNA transduction, and production of transgenic mice, including the study of genomically mosaic effects of transgene integration and/or expression in subsets of cells. In vitro experimentation provides further proof of concept for gencDNA functionality: heterologous expression of in-frame coding sequences were translated into proteins and resulted in decreased cell survival. Additional in vitro experiments recreated gencDNAs in cell lines and identified three conditions required for SGR of APP: 1) APP transcription and splicing, 2) DNA strand breaks, and 3) reverse-transcriptase (RT) activity (Fig. 2). A defined enzymology is currently unclear and will be discussed below in relation to other conditions of intron loss, along with SGR in the immune system. VDJ recombination, which produces the antibody and T-cell receptor diversity that is integral to the adaptive immune systems of all jawed vertebrates (40Carmona L.M. Schatz D.G. New insights into the evolutionary origins of the recombination-activating gene proteins and V(D)J recombination.FEBS J. 2017; 284 (27973733): 1590-160510.1111/febs.13990Crossref PubMed Scopus (34) Google Scholar), was the first confirmed type of SGR and exclusively operates in B and T cells (36Schatz D.G. Swanson P.C. V(D)J recombination: mechanisms of initiation.Annu. Rev. Genet. 2011; 45 (21854230): 167-20210.1146/annurev-genet-110410-132552Crossref PubMed Scopus (287) Google Scholar, 41Schatz D.G. Ji Y. Recombination centres and the orchestration of V(D)J recombination.Nat. Rev. Immunol. 2011; 11 (21394103): 251-26310.1038/nri2941Crossref PubMed Scopus (303) Google Scholar, 42Schatz D.G. Baltimore D. 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