Download a higher level of chromatin structure.

UBAIII Biologia Molecular
1º Ano
2013/2014
Sumário:
Capítulo X. O núcleo eucariota e o controlo da
expressão genética


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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
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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
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Cromatina
Conjunto de DNA e Histonas
Nucleosomas


5
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Tipos de histonas
Octâmeros de histonas
Histona linker H1
Outras variantes
Ligações com outras moléculas

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6
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Níveis superiores de empacotamento
7
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Super enrolamento
Topoisomerase II
8
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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
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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
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Exemplos de proteínas que se ligam a
histonas

As alterações de alguns resíduos das histonas levava:

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11
Ligação de proteínas específicas
Alteração da interacção entre histonas
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Todas as histonas H4 acetiladas estão a verde.
Conclusão?
12
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29/Nov/2012
Cariotipagem
13
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Telómeros
TTAGGG
AATCCC
Repetidas 500 a 5000 vezes
14
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Problemas na replicação de elementos
lineares
15
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Modificação da extremidade
16
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Telomerase-Uma transcriptase reversa
Telomerases não estão sempre
activas (expressas).
Menor expressãoenvelhecimento e
apoptose
Expressão aumentada 
possibilidade da formação de
tumores.
17
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Centrómeros
São heterocromatina constitutiva ou facultativa?
171pbs  500kbases
Têm proteínas associadas
(H3 é CENP-A ligação dos MT)
18
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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
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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
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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
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
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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

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
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

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
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


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

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

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

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
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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