* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download a higher level of chromatin structure.
Gene expression profiling wikipedia , lookup
Genomic imprinting wikipedia , lookup
Molecular cloning wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Community fingerprinting wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Gene regulatory network wikipedia , lookup
List of types of proteins wikipedia , lookup
Non-coding DNA wikipedia , lookup
Molecular evolution wikipedia , lookup
Gene expression wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Histone acetylation and deacetylation wikipedia , lookup
UBAIII Biologia Molecular 1º Ano 2013/2014 Sumário: Capítulo X. O núcleo eucariota e o controlo da expressão genética 2 Comossomas e cromatina Epigenética Organização do núcleo MJC-T09 29/Nov/2012 Expressão genética O que é? Todas as células do nosso organismo têm o mesmo genoma? Todas têm a mesma expressão genética? 3 MJC-T09 29/Nov/2012 Cromossomas Molécula única de DNA 2 metros de comprimento no total 5cm/cromossoma Têm de estar acessíveis a interação com proteínas transcriptoras e replicadoras; Separados fisicamente uns dos outros sem se “enlearem” Aparecem e desaparecem dependendo da fase do ciclo celular. 4 MJC-T09 29/Nov/2012 Cromatina Conjunto de DNA e Histonas Nucleosomas 5 MJC-T09 29/Nov/2012 Tipos de histonas Octâmeros de histonas Histona linker H1 Outras variantes Ligações com outras moléculas 6 MJC-T09 29/Nov/2012 Níveis superiores de empacotamento 7 MJC-T09 29/Nov/2012 Super enrolamento Topoisomerase II 8 MJC-T09 29/Nov/2012 Heterocromatina e eucromatina Vizualização Vs. Transcrição Heterocromatina constitutiva: telómeros e centrómeros efeito de posição sequências bloqueadoras ou Heterocromatina facultativa: Cromossoma X das fêmeas (ambos estão activos durante a oogenese) mosaicismo em humanos (daltonismo) 9 MJC-T09 29/Nov/2012 Formação de heterocromatina Depende da modificação específica das histomas O código das histonas Acetilação (lisina) “liga” Metilação (lisina ou arginina) “desliga” Fosforilação (serina) Lisinas acetiladas abundantes na H3 da eucromatina As mesmas lisinas metiladas em heterocromatina. 10 MJC-T09 29/Nov/2012 Exemplos de proteínas que se ligam a histonas As alterações de alguns resíduos das histonas levava: 11 Ligação de proteínas específicas Alteração da interacção entre histonas MJC-T09 29/Nov/2012 Todas as histonas H4 acetiladas estão a verde. Conclusão? 12 MJC-T09 29/Nov/2012 Cariotipagem 13 MJC-T09 29/Nov/2012 Telómeros TTAGGG AATCCC Repetidas 500 a 5000 vezes 14 MJC-T09 29/Nov/2012 Problemas na replicação de elementos lineares 15 MJC-T09 29/Nov/2012 Modificação da extremidade 16 MJC-T09 29/Nov/2012 Telomerase-Uma transcriptase reversa Telomerases não estão sempre activas (expressas). Menor expressãoenvelhecimento e apoptose Expressão aumentada possibilidade da formação de tumores. 17 MJC-T09 29/Nov/2012 Centrómeros São heterocromatina constitutiva ou facultativa? 171pbs 500kbases Têm proteínas associadas (H3 é CENP-A ligação dos MT) 18 MJC-T09 29/Nov/2012 Epigenética Nem sempre a hereditariedade depende da sequência de DNA. Há características determinadas epigeneticamente (por associação a proteínas específicas). Ex: inactivação dos cromossomas X das fêmeas Mecanismos epigenéticos normalmente podem reverter. Mecanismos epigenéticos muito associados a histonas 19 São herdadas aleatoriamente As que são sintetizadas de novo têm de ser “codificadas” com as acetilações, fosforilações e metilações correctas. MJC-T09 29/Nov/2012 Núcleo eucariota não é um saco! É um organelo organizado 20 As fibras de cromatina estão organizadas num domínio específico dentro do núcleo MJC-T09 29/Nov/2012 Organização do núcleo eucariota Dirigida pelas proteínas do envelope nuclear. A transcrição ocorre em zonas específicas. Genes envolvidos nos mesmos processos mas estão localizados em cromossomas diferentes são muitas vezes transcritos ao mesmo tempo e interagem 21 MJC-T09 29/Nov/2012 Recursos utilizados Capítulo 6 Karp 6ª Edição. Secção 6.1 Capítulo 12 Karp 4 e 5ª Edição. Secção 12.1 Capítulos 7 e 8 do Biologia Celular e Molecular. Azevedo e Sunkel. 22 MJC-T09 29/Nov/2012 Sumário: Controlo ao nível da transcrição O papel dos factores de transcrição como reguladores da transcrição. Estrutura dos factores de transcrição MJC-T12 12/dez/2013 Control of Gene Expression in Eukaryotes Chromosomes and Chromatin DNA and histones are organized into repeating subunits called nucleosomes. Each nucleosome includes a core particle of supercoiled DNA and histone H1 serving as a linker. DNA is wrapped around the core complex. The histone core complex consists of two molecules each of H2A, H2B, H3, and H4 forming an octamer. H3 H2B H2A H2B H4 H3 © 2013 John Wiley & Sons, Inc. All rights reserved. Nucleosomal organization of chromatin: Schematic diagram (top) and EM of Drosophila cell nucleus with nucleosomes along DNA strand (bottom) Control of Gene Expression in Eukaryotes Chromosomes and Chromatin DNA and histones are organized into repeating subunits called nucleosomes. Each nucleosome includes a core particle of supercoiled DNA and histone H1 serving as a linker. DNA is wrapped around the core complex. The histone core complex consists of two molecules each of H2A, H2B, H3, and H4 forming an octamer. 3D structure of a nucleosome from X-ray crystallography. Core particle at two views (top) and schematic of half of a core particle (side) © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Chromosomes and Chromatin Histone modification is one mechanism to alter the character of nucleosomes. DNA and histones are held together by noncovalent bonds. Ionic bonds between negatively charged phosphates of the DNA backbone and positively charged residues of the histones. Histones, regulatory proteins, and enzymes dynamically mediate DNA transcription, compaction, replication, recombination, and repair. © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Higher Levels of Chromatin Structure Higher Levels of Chromatin Structure A 30-nm filament is another level of chromatin packaging, maintained by histone H1. Chromatin filaments are organized into large supercoiled loops. The presence of loops in chromatin can be seen: In mitotic chromosomes form which histones have been extracted. In meiotic lampbrush chromosomes from amphibian oocytes. 30-nm fiber: EM of a fiber (left) and two packaging models (middle, right). © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Higher Levels of Chromatin Structure Higher Levels of Chromatin Structure A 30-nm filament is another level of chromatin packaging, maintained by histone H1. Chromatin filaments are organized into large supercoiled loops. The presence of loops in chromatin can be seen: In mitotic chromosomes form which histones have been extracted. In meiotic lampbrush chromosomes from amphibian oocytes. Chromatin loops: a higher level of chromatin structure. EM: of a mitotic chromosome (left) and model for cohesin in maintaining loops (right) © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Higher Levels of Chromatin Structure Higher Levels of Chromatin Structure A nucleus 10 mm in diameter can pack 200,000 times this length of DNA within its boundaries. Packing ratio of the DNA in nucleosomes is approximately 7:1. Assembly of the 30-nm fiber increases the DNApacking ratio to 40:1. Mitotic chromosomes represent the ultimate in chromatin compactness with a ratio of 10,000:1. © 2013 John Wiley & Sons, Inc. All rights reserved. Levels of organization of chromatin. Control of Gene Expression in Eukaryotes Heterochromatin and Euchromatin Heterochromatin and Euchromatin Euchromatin returns to a dispersed state after mitosis. Heterochromatin is condensed during interphase. Constitutive heterochromatin remains condensed all the time. Found mostly around centromeres and telomeres. Consists of highly repeated sequences and few genes. Facultative heterochromatin is inactivated during certain phases of the organism’s life. Is found in one of the X chromosomes as a Barr body through X inactivation. X inactivation is a random process, making adult females genetic mosaics. © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Heterochromatin and Euchromatin Inactivated X chromosome or Barr body (arrows). Calico cat cloning: Random inactivation of the X chromosome in different cells during embryonic development creates a mosaic of tissue patches. • Facultative heterochromatin is inactivated during certain phases of the organism’s life. – Is found in one of the X chromosomes as a Barr body through X inactivation. – X inactivation is a random process, making adult females genetic mosaics. © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes The histone code The “histone code.” Histones can be modified by addition of methyl, acetyl, and phosphate groups The Histone Code and Formation of Heterochromatin The histone code hypothesis states that the activity of a chromatin region depends on the degree of chemical modification of histone tails. Histone tail modifications influence chromatin in two ways: Serve as docking sites to recruit nonhistone proteins. Alter the way histones of neighboring nucleosomes interact. © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes The histone code Proteins that bind selectively to modified H3 or H4 residues The majority of modified amino acids reside on the N-termini of H3 and H4. Each of the bound proteins possesses an activity that alters the structure and/or function of the chromatin. Heterochromatin has many methylated H3 histones, which stabilize the compact nature of the chromatin. © 2013 John Wileyplay & Sons, All rights Small RNAs and specific enzymes a Inc. role in histone methylation. reserved. Control of Gene Expression in Eukaryotes Histone modification Removal of the acetyl groups from H3 and H4 histones is among the initial steps in conversion of euchromatin into heterochromatin. Histone deacetylation is accompanied by methylation of H3K9 histone methyltransferase (SUV39H1 in humans. Methylated H3K9 binds to proteins with a chromodomain, for example heterochromatic protein 1 (HP1) Once HP1 is bound to the histone tails, HP1-HP1 interactions facilitate chromatin packaging into a heterochromatin state, Correlation between transcriptional activity and histone acetylation. Chromosomes labeled with fluorescent antibodies to acetylated histone H4 stain all chromosomes except the inactivated X (arrow). © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Histone modification Removal of the acetyl groups from H3 and H4 histones is among the initial steps in conversion of euchromatin into heterochromatin. Histone deacetylation is accompanied by methylation of H3K9 histone methyltransferase (SUV39H1 in humans. Methylated H3K9 binds to proteins with a chromodomain, for example heterochromatic protein 1 (HP1) Once HP1 is bound to the histone tails, HP1-HP1 interactions facilitate chromatin packaging into a heterochromatin state, Histone deacetylase Histone methyltransferase Model of possible events during the formation of heterochromatin © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes The Structure of a Mitotic Chromosome The Structure of a Mitotic Chromosome Chromatin of a mitotic cell exists in its most highly condensed state. Staining mitotic chromosomes can provide useful information. A karyotype is a preparation of homologous pairs ordered according to size. The pattern on a karyotype may be used to screen chromosomal abnormalities. Procedure to prepare mitotic chromosomes for microscopic observation from leukocytes © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes The Structure of a Mitotic Chromosome The Structure of a Mitotic Chromosome Chromatin of a mitotic cell exists in its most highly condensed state. Staining mitotic chromosomes can provide useful information. A karyotype is a preparation of homologous pairs ordered according to size. The pattern on a karyotype may be used to screen chromosomal abnormalities. © 2013 John Wiley & Sons, Inc. All rights reserved. Human mitotic chromosomes labeled with different specific fluorescent dyes. The stained chromosomes of a human male arranged in a karyotype Control of Gene Expression in Eukaryotes Telomeres Telomeres The end of each chromosome is called a telomere and is distinguished by a set of repeated sequences. New repeats are added by a telomerase, a reverse transcriptase that synthesizes DNA from a DNA template. Telomeres are required for the complete replication of the chromosome because they protect the ends from being degraded. Telomerase activity is thought to have major effects on cell life. © 2013 John Wiley & Sons, Inc. All rights reserved. In situ hybridization with a DNA probe (TTAGGG) to locate telomeres on human chromosome Proteins can bind to telomeres: RAP1 in yellow, DNA in blue Control of Gene Expression in Eukaryotes Telomeres Telomeres The end of each chromosome is called a telomere and is distinguished by a set of repeated sequences. New repeats are added by a telomerase, a reverse transcriptase that synthesizes DNA from a DNA template. Telomeres are required for the complete replication of the chromosome because they protect the ends from being degraded. Telomerase activity is thought to have major effects on cell life. The end-replication problem: Generation of single stranded overhangs that shorten DNA © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Telomeres Telomeres The end of each chromosome is called a telomere and is distinguished by a set of repeated sequences. New repeats are added by a telomerase, a reverse transcriptase that synthesizes DNA from a DNA template. Telomeres are required for the complete replication of the chromosome because they protect the ends from being degraded. Telomerase activity is thought to have major effects on cell life. The single-stranded overhang is not free but forms a loop. The loop is a binding site for telomere-capping proteins that protect the ends of the chromosomes and regulate telomere length. © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Telomeres Telomeres The end of each chromosome is called a telomere and is distinguished by a set of repeated sequences. New repeats are added by a telomerase, a reverse transcriptase that synthesizes DNA from a DNA template. Telomeres are required for the complete replication of the chromosome because they protect the ends from being degraded. Telomerase activity is thought to have major effects on cell life. The mechanism of action of telomerase. Gap in complementary strand filled by DNA polymerase a (carries © 2013 John Wiley & Sons, Inc. All rights DNA primer). reserved. Control of Gene Expression in Eukaryotes Telomeres Telomeres The end of each chromosome is called a telomere and is distinguished by a set of repeated sequences. New repeats are added by a telomerase, a reverse transcriptase that synthesizes DNA from a DNA template. Telomeres are required for the complete replication of the chromosome because they protect the ends from being degraded. Telomerase activity is thought to have major effects on cell life. The importance of telomerase in maintaining chromosome integrity. Chromosomes from a telomerase knockout mouse cell shows some chromosomes lack telomeres entirely (stained yellow) and some have fused to one another at their ends © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Telomeres Telomeres In somatic cells, telomere lengths are reduced each cell division to limit cell doublings. A critical point occurs from telomere shortening when cells stop their growth and division. In contrast, cells that are able to resume telomerase expression continue to proliferate. These cells continue to divide and do not shown normal signs of aging. Approximately 90% of human tumors have cells with active telomerase. Telomerase dynamics during normal and abnormal growth. Limited telomerase levels in somatic cells reduces the amount of cell doublings compared to germ cells, unless telomerase is reactivated. © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Centromeres Scanning electron micrograph of a mitotic chromosome with the centromere marked by a distinct indentation. Centromeres The centromere is located at the site markedly indented on a chromosome. Centromeres contain constitutive heterochromatin. Centromeric DNA is the site of microtubule attachment during mitosis. DNA sequence is not important for centromere structure and function. Histone H3 variant CENP-A is found in the centromeres to potentially function in kinetochore assembly. © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Epigenetics Epigenetics: There’s More to Inheritance than DNA Epigenetic inheritance depends on factors other than DNA sequences. X-chromosome inactivation is an example, since the two X chromosomes can have identical DNA sequences, but one is inactivated and the other is not. An epigenetic state can usually be reversed; X chromosomes, for example, are reactivated prior to formation of gametes. Differences in disease susceptibility and longevity between genetically identical twins may be due, in part, to epigenetic differences that appear between the twins as they age. Parental histones determine the chemical modifications found in the newly synthesized histones. As heterochromatin is replicated, a histone methyltransferase labels the newly synthesized H3 molecules added into the daughter nucleosomes. Euchromatic regions tend to contain acetylated H3 tails, a modification transmitted from parental chromatin to progeny chromatin. © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Nuclear organization 3D map of all of the chromosomes present in a human fibroblast nucleus. Each chromosome, represented as an identifiable color, is found to occupy a distinct territory within the nucleus. The Nucleus as an Organized Organelle Chromatin fibers of an interphase chromosome are not diffuse and random, but are concentrated into distinct territories. Genes are physically moved to nuclear sites called transcription factories where transcription machinery is located (e.g., hormone induction). DNA sequences that participate in a common biological response but reside on different chromosomes interact within the nucleus. © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Nuclear organization Localizing specific chromosomes within an interphase nucleus. More active chromosomes, those that have more protein-coding genes, are centrally located in the nucleus. The Nucleus as an Organized Organelle Chromatin fibers of an interphase chromosome are not diffuse and random, but are concentrated into distinct territories. Genes are physically moved to nuclear sites called transcription factories where transcription machinery is located (e.g., hormone induction). DNA sequences that participate in a common biological response but reside on different chromosomes interact within the nucleus. © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Nuclear organization Breast cancer cells treated with estrogen co-activate genes on Chr2 and Chr21 Model of how different DNA regions could be organized for gene expression The Nucleus as an Organized Organelle Chromatin fibers of an interphase chromosome are not diffuse and random, but are concentrated into distinct territories. Genes are physically moved to nuclear sites called transcription factories where transcription machinery is located (e.g. hormone induction). DNA sequences that participate in a common biological response but reside on different chromosomes interact within the nucleus. © 2013 John Wiley & Sons, Inc. All rights reserved. Control of Gene Expression in Eukaryotes Nuclear organization Antibody staining against an mRNA processing factor shows 30-50 distinct sites Time course of viral gene expression in infected cells showing splicing factors (orange) compared to integration site (white arrow) The Nucleus as an Organized Organelle Chromatin fibers of an interphase chromosome are not diffuse and random, but are concentrated into distinct territories. Genes are physically moved to nuclear sites called transcription factories where transcription machinery is located (e.g. hormone induction). DNA sequences that participate in a common biological response but reside on different chromosomes interact within the nucleus. © 2013 John Wiley & Sons, Inc. All rights reserved. The Human Perspective: Chromosomal Aberrations and Human Disorders A chromosomal aberration is loss or exchange of a segment between different chromosomes, caused by exposure to DNA-damaging agents. Chromosomal aberrations have different consequences depending on whether they are in somatic or germ cells. Inversions involve the breakage of a chromosome and resealing of the segment in a reverse order. Translocations are the result of the attachment of all or one piece of one chromosome to another chromosome. Translocation. Exchange between chr12 (bright blue) and chr7 (red) in human cells © 2013 John Wiley & Sons, Inc. All rights reserved. The effect of inversion. Crossing over between a normal chromosome (purple) and one containing an inversion (green) The Human Perspective: Chromosomal Aberrations and Human Disorders A chromosomal aberration is loss or exchange of a segment between different chromosomes, caused by exposure to DNAdamaging agents. Chromosomal aberrations have different consequences depending on whether they are in somatic or germ cells. Deletions result when there is loss of a portion of a chromosome. Duplications occur when a portion of a chromosome is repeated. Translocation and evolution. If the only two ape chromosomes that have no counterpart in humans are hypothetically fused, they match human chromosome number 2, band for band. © 2013 John Wiley & Sons, Inc. All rights reserved. Recursos utilizados Capítulo 6 Karp 7ª Edição. Secção 6.4 ou 12.4 Capítulo 12 Karp 4 e 5ª Edição. Secção 12.4 MJC-T12 12/dez/2013