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GENERAL VIEW OF THE HUMAN GENOME
Gene landscape
• In prokaryotes, genes are often tandemly arranged, with little or no spacer sequences in between
• In eukaryotes, there is considerable spacer DNA between genes– some is repetitive DNA: identical or nearly identical
repeated units (much is derived from mobile genetic elements)
– …but also are unique sequences
GENERAL VIEW OF THE HUMAN GENOME
Segmental duplications (low-copy repeats)
•Large block (1 >200Kb)•Highly Similar (>97%)•Without characteristic sequences
A A’
3) Segmental duplications
Closely related sequence blocks at different genomic loci
Transfer of 1-200kb blocks of genomic sequence
Segmental duplications can occur on homologous chromosomes (intrachromosomal) or non homologous chromosomes (interchromosomal)
Not always tandemly arranged Relatively recent (population polymorphisms) Found especially around centromeres and
telomeres
Segmental duplications
Interchromosomal segments duplicated
among non-homologous
chromosomes
Intrachromosomal duplications occur within a
chromosome / arm
Nature Reviews Genetics 2, 791-800 (2001);
Duplications (and rearrangements) are very common:intrachromosomal
HUMPHRAY et al. (2004), Nature 429, 369-374
SEGMENTAL DUPLICATIONS ON CHROMOSOME 9:IINTERCHROMOSOMAL DUPLICATIONS LARGE DUPLICATIONS (GREATER THAN10 kb) ARE SHOWN WITH CHROMOSOME9 MAGNIFIED; OTHER CHROMOSOMESARE ARRANGED IN THE ORDER IN WHICHTHEY MATCH CHROMOSOME (GRAYRECTANGLE IS HETEROCHROMATIN)
CNVs IN POPULATIONS
CNVs AND DISEASE
Recent segmental duplications are polymorphic
GENERAL VIEW OF THE HUMAN GENOME
Tandemly repetitive DNA
Class size of repeat locations
satellite DNA (100 kb to several Mb)
5bp - 5 kb heterochromatin particularly at centromeres
Minisatellite DNA 0.1 - 20kb
6 - 64 bp scattered but concentrated near telomeres
Microsatellite DNA <100bp
1 - 4 bp dispersed throughout genome
(SSRs)
(about 3% of Human Genome)
Variable Nucleotide Tandem Repeats
(VNTRs)
• VNTRs are short segments of DNA that repeat a few to hundreds of times
• These unusual repeats occurs many different spots on human chromosomes
• Each individual will have different numbers of these VNTRs at each of these spots
• Each of these spots, or loci, are given different names (MSRs, STRs, AmpFLPs, etc)
• VNTRs are inheritable – the numbers of repeats at each location in you are a random combination of the VNTRs in your parents
Practical Applications with VNTR’s1. Genetic Identification Services (GIS) was established in 1994 to serve the agriculture, aquaculture, and wildlife ecology communities through the development and use of custom genetic markers for a wide variety of applications. GIS has developed libraries for academic, commercial, and government entities. GIS has also developed a data base of loci in studies involving a wide range of plant and animal species.
EXAMPLES OF GIS APPLICATIONS Identification of economically important genetic traits Selective breeding Population structure and dynamics Monitoring genetic diversity Wildlife forensics Proprietary stock and strain protection Parent-offspring identification Sexing
PROBLEMS with determining a reasonable VNTR “match”
VNTRs, are not distributed evenly across all of human population. A given VNTR cannot, therefore, have a stable probability of occurrence; it will vary depending on an individual's genetic background.
The difference in probabilities is particularly visible across racial lines. Some VNTRs that occur very frequently among Hispanics will occur very rarely among Caucasians or African-Americans. Additionally, the heterogeneous genetic composition of interracial individuals, who are growing in number, presents an entirely new set of questions.
This type of population genetics has been hindered by controversy, because the idea of identifying people through genetic anomalies along racial lines comes alarmingly close to the eugenics and ethnic purification movements
and, some argue, could provide a scientific basis for racial discrimination.
GENERAL VIEW OF THE HUMAN GENOME
Tandemly repetitive DNA
Class size of repeat locations
satellite DNA (100 kb to several Mb)
5bp - 5 kb heterochromatin particularly at centromeres
Minisatellite DNA 0.1 - 20kb
6 - 64 bp scattered but concentrated near telomeres
Microsatellite DNA <100bp
1 - 4 bp dispersed throughout genome
Human telomeres: (TTAGGG)n / variable total length
Satellite DNA
Chromosomal location of tandemly repetitive DNA
GENERAL VIEW OF THE HUMAN GENOME
32
Repetitive DNA
Iinterspersedinterspersed in tandemin tandem
TEsTEs are like that!are like that!
Transposable elements in the human genome
RNA or DNA intermediate
• Transposon moves using DNA intermediate
• Retrotransposon moves using RNA intermediate
In 100,000 bp of human DNA:Genes 1 DNA transposons 10Simple Satellite 30 Viral retroposons 15Mini Satellite 1 Lines (L1) 25Micro Satellite 10 Sines (Alu) 50
Repeats and Evolution
• Recombination Hotspots• Unequal Crossover• Transposition• Evolutionary History• Exon Shuffl ing
2
Transposable element (transposon)A discrete segment of DNA able to move to a new site in the genome
(without any homology to the target locus)
Transposable element (transposon)A discrete segment of DNA able to move to a new site in the genome
(without any homology to the target locus)
Transposons and Transposition
TranspositionMovement of a transposable element to a new site in the genome
3
Consequences of Transposition. Part 1
gene disruption
wild-type gene product
REG TE
altered gene expression
TE
no gene product
changes in gene expression
wild-type gene expression
REGREG
4
Consequences of Transposition. Part 2
a b cTE TE abcTE TE
deletion
TE
crossing over
TE
abc
TE
pairing
inversion
cba TETE
crossing over
a
b
TE
TE
c
pairing
5
Consequences of Transposition. Part 3
crossing over
reciprocal chromosome translocation
TE
TE
TE
TE
crossing over
acentric and dicentric chromosomes
TE
TE
TE
TE
Repeats and Evolution
• Recombination Hotspots• Unequal Crossover• Transposition• Evolutionary History• Exon Shuffl ing
transposase gene
IR IR
transpositionDNA break
repair of double-strand break, using sister chromatid
DNA TE
Transposable elements in the human genome
Reverse transcryptase
Integrase
RNA polymerase II
DNA
RNA
RNA
Short interspersed repetitive elements: SINEs
• Example: Alu repeats– Most abundant repeated DNA in primates
– Short, about 300 bp
– About 1 million copies
– Likely derived from the gene for 7SL RNA
– Cause new mutations in humans
• They are retrotranposons– DNA segments that move via an RNA intermediate.
• MIRs: Mammalian interspersed repeats– SINES found in all mammals
• Analogous short retrotransposons found in genomes of all vertebrates.
31
AluAlu elementselements Length = ~300 Length = ~300 bpbp
Repetitive: > 1,000,000 times in the human genomeRepetitive: > 1,000,000 times in the human genome
Constitute >10% of the human genome Constitute >10% of the human genome
Derive Derive from 7SL RNAfrom 7SL RNA
Found mostly in Found mostly in intergenicintergenic regions and regions and intronsintrons
Propagate in the genome through Propagate in the genome through retropositionretroposition (RNA (RNA intermediates). intermediates).
33
Evolution of Evolution of AluAlu elementselements
35
AluAlu elements can be divided into elements can be divided into subfamiliessubfamilies
The subfamilies are The subfamilies are distinguished by distinguished by ~16 diagnostic ~16 diagnostic positions.positions.
RNA pol III promoter
RNA pol III
Alu"שלוהפרומוטר יחד עם "קופץ
RNA
DNA
Can cause disease when they insert into an essential gene. Examples:
Neurofibromatosis
Huntington’s Disease
Some forms of inherited breast cancer
Alu insertions
LDL receptor gene
Alu repeats present within introns
Alu repeats in exons
4
4
4
5
5
5 6
6
6
Alu Alu
AluAlu
X
4 6Alu
unequalcrossing over
one product has adeleted exon 5
(the other product is not shown)
BRCA1
Alu repeats present within introns (40%)
Alu repeats in exons
Alu
5’UTR
Normal tissue
(10 times lower efficiency of translation)
Breast cancertissue
Other SINEs:
SVA elements
Long interspersed repetitive elements: LINEs
• Moderately abundant, long repeats– L1 family: most abundant– Up to 7000 bp long
• Retrotransposons– Encode reverse transcriptase and other enzymes
required for transposition– No long terminal repeats (LTRs)
• Homologous repeats found in all mammals and many other animals
• Some actively transposing (cause new mutations in humans)
• Containing pol II promoter and two ORFs
DNA:RNA hybrid
reverse transcription
DNA “2nd strand” synthesis
integration at new position in DNAoriginal copy still there
retrotransposon in DNA
Events in L1 transposition
ORF1
ORF2RT’aseendonuclease
3’ UTRpromoter
FDR FDR
transcribe
Staggered break at target
Priming of synthesis by RT’ase at staggered breakPriming of synthesis by RT’ase at staggered break
2nd strand synthesis and repair of staggered break
RT’ase works preferentially on L1 mRNA
Gene A
Gene B
. Relative positions of the apolipoprotein(a) [apo(a)] gene and its enhancer element. The long interspersed nuclear element (LINE) (green) is located approximately 20 kilobases upstream from the start of transcription of the apo(a) gene (blue). The enhancer element (red) is located entirely within the LINE element and is competent to enhance expression levels of apo(a). Tissue specificity is provided by the core promoter element (yellow).
Modified, with permission, from [28].
AUMENTO DE LA CAPACIDAD INFORMACIONAL
GENOMA PROTEOMA
DUPLICACIONESGÉNICASSEGMENTALESPOLIPLOIDÍA
CAMBIOS EN REGIONESREGULADORAS
IMPORTACIÓN DEGENES DE OTROS ORGANISMOS
DISTINTOS PROMOTORESSEÑALES DE poli(A)SPLICING ALTERNATIVOEDITINGMODIFICACIONES POSTRADUCCIONALES …
(SÓLO SE MUESTRANALGUNOS MECANISMOS)
TRANSFERENCIA HORIZONTAL
ENDOSIMBIOSIS
BACTERIANARETROVIRAL
TRANSFERENCIA PARCIAL(COMÚN EN PROCARIONTES)
EL SISTEMA FOTOSINTÉTICOEL SISTEMA INMUNE (COMBINATORIO)
TRANSFERENCIA HORIZONTAL DE GENOMAS COMPLETOS
(ENDOSIMBIOSIS)
BACTERIANA (EL/LOS ORIGEN/ES DE LA CÉLULA EUCARIONTE)
RETROVIRAL(TODOS LOS GENOMAS MULTICELULARES)
Gag- cápsida (CA)-matriz (MA)
- Nucleocápsida (NC)
Pol-proteasa (PR)-Integrasa (IN)
-Transcriptasa Inversa (RT)
RNA (diploide)
Membrana lipídica
EnvSuperficie (SU)
Transmembrana (TM)
gag pol envR
U5 RU3
Retrotranscripción
gag pol envU3 R U5 U3 R U5
LTR LTR
Proteínasestructurales
Enzimas Proteínas decubierta
RNA (PARTÍCULA VIRAL)
DNA (PROVIRUS INTEGRADO)
Gag- cápsida (CA)-matriz (MA)
- Nucleocápsida (NC)
Pol-proteasa (PR)-Integrasa (IN)
-Transcriptasa Inversa (RT)
RNA (diploide)
Membrana lipídica
EnvSuperficie (SU)
Transmembrana (TM)
RECEPTORCELULAR
ssRNA dsDNA
Retrotranscripción
INTEGRACIÓNEN EL GENOMA
TRANSCRIPCIÓNmRNAsProteínas
virales
+RNA viral no procesadoR-U5-gag-pol-env-U3-R
?
PARTÍCULA VIRAL (INFECTIVA)
CÉLULAS SOMÁTICAS
REPLICACIÓNVIRAL
INTEGRACIÓNVIRAL
DIVISIONES MITÓTICAS
¿?
CÉLULAS GERMINALES
INTEGRACIÓN VIRAL(ENDOGENIZACIÓN)
TRANSMISIÓN A LA DESCENDENCIA(HERENCIA MENDELIANA)
INFECCIÓN AOTROS INDIVIDUOS(TRANSMISIÓNHORIZONTAL)
RETROVIRUS ENDÓGENOSRETROVIRUS
EXÓGENOS
¿POR QUÉ SE MANTIENEN EN LOS GENOMAS?
PARASITISMO: SU ÚNICA FUNCIÓN ES AUTOPERPETUARSEEN COMPETENCIA CON LAS SECUENCIAS CELULARES
(DNA EGOÍSTA O DNA BASURA)
SIMBIONTES: UNA VEZ INTEGRADOS, ALGUNAS DE SUSFUNCIONES SON APROVECHADAS POR LA CÉLULA
(EXAPTACIÓN O DOMESTICACIÓN)
CONFORMAN LOS GENOMAS Y FUNCIONAN ENCOORDINACIÓN CON EL RESTO DE LAS SECUENCIAS
(SON NECESARIAS PARA LOS ORGANISMOS)
SON UN MECANISMO PRINCIPAL DE CAMBIO GENÓMICOSENSIBLE A LOS CAMBIOS AMBIENTALES
(SON NECESARIOS PARA LA EVOLUCIÓN ORGÁNICA)
ALTERNATIVA:
LOS RETROVIRUS ENDÓGENOS ESTÁN PRESENTESEN (CASI) TODOS LOS ORGANISMOS EUCARIONTES
SON SECUENCIAS ACTIVAS Y PARTICIPANEN LAS FUNCIONES CELULARES NORMALES
SUS FUNCIONES ESTÁN INTEGRADAS EN LOSPROCESOS FISIOLÓGICOS COMPLEJOS DE LOSORGANISMOS
¿SON NECESARIOS PARA LOS ORGANISMOS?
UNIVERSALIDAD Y ESPECIFICIDAD
INSERCIÓN EN LÍNEA GERMINAL
TRANSMISIÓN VERTICAL
MUTACIÓN RETROTRANSPOSICIÓN RECOMBINACIÓN
NUEVAS COMBINACIONESNUEVOS PROVIRUS
TRANSMISIÓN VERTICALNUEVAS CARACTERÍSTICAS INFECTIVAS
TRANSMISIÓN HORIZONTAL A NUEVOSINDIVIDUOS Y/O ESPECIES
DESCENDENCIA
FUNCIONES CELULARES DE LOS RETROVIRUS ENDÓGENOS
GENES ESTRUCTURALESgag
env
EstructuralesUnión específica a DNA/RNA
PROTEÍNAS
GENES DE ENZIMASPro
pol
RetrotranscriptasaRnasa HIntegrasadUTPasa*
LTRs Funciones Reguladoras
Promotores/EnhancersProcesamiento de RNAs(Señales de splicing ypoliadenilación)
Inmunomoduladorasfusogénicas
LTRs
HORMONAS(y otros…)
FACTORES DETRANSCRIPCIÓN
RNA polimerasa II
PROMOTORES ESPECÍFICOSDE TIPO CELULAR QUE RESPONDEN A SEÑALES
INTERNAS Y EXTERNAS A LA CÉLULA
GENES PROVIRALES OTROS GENES
(MILES DE LTRs SOLITARIASDISPERSAS POR EL GENOMA)
LTRs
SEÑALES DE SPLICING SEÑALES DE POLIADENILACIÓN
SPLICEOSOME POLI-A-POLIMERASA
•SPLICING ALTERNATIVO•SPLICING INTERGÉNICO
mRNAs QUIMÉRICOS
MÚLTIPLES PRODUCTOS PROTEICOSCON DISTINTOS DOMINIOS Y CON
FUNCIONES DIFERENTES
LOS RETROVIRUS ENDÓGENOS EN LOS PROCESOSFISIOLÓGICOS COMPLEJOS: LA PLACENTACIÓN
PROBLEMAS GENÉTICOS DE LA PLACENTACIÓN:
•EL SISTEMA INMUNOLÓGICO DE LA MADREDEBERÍA RECHAZAR UN EMBRIÓN
“MEDIO EXTRAÑO”
•LA MADRE DEBE CONTROLAR LA INVASIÓN Y EL CRECIMIENTO DEL FETO
•EL SISTEMA INMUNOLÓGICO DE LA MADREDEBERÍA RECHAZAR UN EMBRIÓN
“MEDIO EXTRAÑO”
•LA MADRE DEBE CONTROLAR LA INVASIÓN Y EL CRECIMIENTO DEL FETO
env
INMUNOSUPRESOR
FUSOGÉNICO
PARTÍCULAS VIRALES
INFECCIÓN LOCAL DE MACRÓFAGOS MATERNOS
SINCITIOTROFOBLASTOSINCITINA(HERV-W)
DIFERENCIACIÓN TERMINALDEL TROFOBLASTO INVASIVO
PARTÍCULAS VIRALES
VARIOSHERVs
CONTROL DEL CRECIMIENTO FETAL
INMUNOSUPRESIÓN
INFECCIÓN LOCAL DE MACRÓFAGOS MATERNOS
SINCITIOTROFOBLASTOSINCITINA(HERV-W)
DIFERENCIACIÓN TERMINALDEL TROFOBLASTO INVASIVO
PARTÍCULAS VIRALES
VARIOSHERVs
CONTROL DEL CRECIMIENTO FETAL
INMUNOSUPRESIÓN
LA PLACENTACIÓN ES UN PROCESO COMPLEJOQUE INTEGRA MÚLTIPLES FACTORES
INTERLEUKINAS
FACTORES DE CRECIMIENTO
BALANCE ENERGÉTICO MATERNO-FETAL
OTROS MUCHOS GENES...
LOS VIRUS ACTIVAN LOS GENES DE INTERLEUKINAS
HERV-E-PTN ESPECÍFICO DE PLACENTA
LA LEPTINA PLACENTARIA Y SU RECEPTORSON ISOFORMAS CONTROLADAS POR HERVs
LAS LTRs SON PROMOTORES ALTERNATIVOSESPECÍFICOS DE PLACENTA PARA ALGUNOS GENES (APO-C-I; EBR...)
Nature. 2012 Jul 5;487(7405):57-63.Embryonic stem cell potency fluctuates with endogenous retrovirus activity.Macfarlan TS, Gifford WD, Driscoll S, Lettieri K, Rowe HM, Bonanomi D, Firth A, Singer O, Trono D, Pfaff SL.
Embryonic stem (ES) cells are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass, which lacks the ability to produce all extraembryonic tissues. Here we identify a rare transient cell population within mouse ES and induced pluripotent stem (iPS) cell cultures that expresses high levels of transcripts found in two-cell (2C) embryos in which the blastomeres are totipotent. We genetically tagged these 2C-like ES cells and show that they lack the inner cell mass pluripotency proteins Oct4 (also known as Pou5f1), Sox2 and Nanog, and have acquired the ability to contribute to both embryonic and extraembryonic tissues. We show that nearly all ES cells cycle in and out of this privileged state, which is partially controlled by histone-modifying enzymes. Transcriptome sequencing and bioinformatic analyses showed that many 2C transcripts are initiated from long terminal repeats derived from endogenous retroviruses, suggesting this foreign sequence has helped to drive cell-fate regulation in placental mammals.
LOS RETROVIRUS ENDÓGENOS COMO MECANISMOSDEL CAMBIO GENÓMICO: PLASTICIDAD GENÓMICA
RECOMBINACIÓN
RETROVIRUS ENDÓGENOS
RETROTRANSPOSICIÓN
RETROTRANSFECCIÓN
EFECTOS DE LA RECOMBINACIÓN ENTRE DISTINTASSECUENCIAS RETROVIRALES ENDÓGENAS:
•DUPLICACIÓN GÉNICA (ESTRUCTURAL)
•GANANCIA / PÉRDIDA DE EXONES (i.e. DE DOMINIOS PROTEICOS FUNCIONALES)
•REORDENACIONES CROMOSÓMICAS(i.e. REORGANIZACIONES GENÓMICAS MASIVAS)
EFECTOS DERIVADOS DE LA RETROTRANSPOSICIÓN
AMPLIFICACIÓN MOVILIDAD
•ADQUISICIÓN DE NUEVAS FUNCIONES
•CONFERIR DISTINTA REGULACIÓN A OTRAS SECUENCIAS
•PRODUCCIÓN DE NUEVOS RNAs QUIMÉRICOS
•DIVERSIDAD ALÉLICA EN LAS POBLACIONES (MHC)
•MUTAGÉNESIS INSERCIONAL
INCREMENTO DE LA CAPACIDAD COMBINATORIA
CAPACIDAD DE RETROTRANSFECCIÓN
RNAs
ENCAPSULAMIENTO EN LAPARTÍCULA RETROVIRAL
INFECCIÓN DE LA LÍNEA GERMINAL
RETROGENES RETROPSEUDOGENES
ESTRUCTURALES OENZIMÁTICOS
CODIFICANTES
tRNAssnRNAssnoRNAsstRNAs...
mRNAs
•GERMINALES•SOMÁTICOS
INFECCIÓN DE LA LÍNEA GERMINAL
RETROGENES RETROPSEUDOGENES
tRNAssnRNAssnoRNAsstRNAs...
mRNAs
•GERMINALES•SOMÁTICOS
MULTIPLICACIÓN FUNCIONAL DE SECUENCIASCON CAMBIO DE ENTORNO REGULADOR
INCREMENTO DE LA CAPACIDAD INFORMACIONAL
WEISSMAN
INCREMENTO DE LA CAPACIDAD COMBINATORIA
INCREMENTO DE LA CAPACIDAD INFORMACIONAL
+
GENERACIÓN DE DIVERSIDAD ESTRUCTURAL Y FUNCIONAL
REMODELACIÓN GLOBAL DE LOS GENOMAS
LAS REORGANIZACIONES GENÓMICAS MEDIADASPOR LOS RETROVIRUS ENDÓGENOS DEPENDEN
DE LAS CONDICIONES AMBIENTALES
CONDICIONES EXTERNASCRÍTICAS
SITUACIONES DESHOCK GENÓMICO
TRANSPOSICIÓN CONCERTADADE ELEMENTOS MÓVILES
(ALGUNAS) SITUACIONES DE STRESS AMBIENTAL
•ENDOGAMIA FORZADA•HIBRIDACIÓN INTERESPECÍFICA•CULTIVOS CELULARES PROLONGADOS•AGENTES MUTAGÉNICOS EXTERNOS
-RAYOS UVB
-ANÁLOGOS DE BASES•INFECCIONES VIRALES Y BACTERIANAS•FALLO CELULAR GENERALIZADO (p.e. NEOPLASIA)
DESREGULACIÓN CUANTITATIVA YESPACIO-TEMPORAL DE LOSRETROVIRUS ENDÓGENOS
LOS RETROVIRUS ENDÓGENOS FORMAN PARTEDE LA RESPUESTA CELULAR ANTE SITUACIONESCRÍTICAS DE STRESS INDUCIDO
CONDICIONESAMBIENTALESADVERSAS
AUMENTO DEACTIVIDAD
CAMBIOS GENÓMICOSEXTENSIVOS
FORMACIÓN DE PARTÍCULAS VIRALES
EXTENSIÓN A LA POBLACIÓN DE NUEVASCOMBINACIONES O SALTO DE ESPECIE
CAMBIO GENÓMICO INDUCIDO POR LAS CONDICIONES AMBIENTALES Y MEDIADA POR LOS
RETROVIRUS ENDÓGENOS
LAS TASAS DE CAMBIO GENÓMICO NO SON CONSTANTES
ANTE SITUACIONES AMBIENTALES CRÍTICAS SE PRODUCEN EXPLOSIONES RETROTRANSPOSICIONALES
QUE IMPLICAN REORGANIZACIONES GENÓMICASGLOBALES
APARECEN NUEVAS ORGANIZACIONESMORFO-FISIOLÓGICAS
DISCONTINUIDADES EVOLUTIVAS
LOS CAMBIOS GENÓMICOS PROVOCADOSPOR LOS RETROVIRUS ENDÓGENOS:
NO SON GRADUALES: NO TIENEN UNA TASACONSTANTE SINO QUE SE ALTERNAN ETAPASDE REMODELACIÓN EXTENSIVA CON OTRASDE (RELATIVA) ESTABILIDAD
NO SUCEDEN AL AZAR, SINO DE MANERACOORDINADA Y EN RESPUESTA A LASCONDICIONES AMBIENTALES
ALGUNAS IMPLICACIONES PRÁCTICAS:
•TRATAMIENTOS ANTI-SIDA
•LOS RETROVIRUS COMO VECTORESDE TERAPIA GÉNICA
•XENOTRASPLANTES
