Accessing the Genome

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Accessing the Genome
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Outline

• 8.1. Inside the Nucleus
• 8.2. Chromatin Modifications and Genome
  Expression

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Inside the Nucleus

• The internal architecture of the eukaryotic
  nucleus
• Chromatin domains

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The internal architecture of the eukaryotic
nucleus(1/3)

• Two novel types of microscopy analysis
• Electron microscopy
• Fluorescent microscopy

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The internal architecture of the eukaryotic
nucleus(2/3)

• Electron microscopy

 image analysis3D restructuring of
images          three types of electron
microscopy                TEM  - 
Transmission               SEM  -  Scanning  
                 FEM  -  Freeze Fracture  
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                                                          Only, killed, fixed, and sectioned specimens          (no living cells) may be viewed. Lens are electromagnets that focus high velocity e's
                                                                                                                                                      
                                                                                                                                 
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FEM
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FEM  -  Freeze Fracture
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FEM  -  Freeze Fracture
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The internal architecture of the eukaryotic
nucleus(1/3)

• Two novel types of microscopy analysis
• Electron microscopy
• Fluorescent microscopy

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The internal architecture of the eukaryotic
nucleus(3/3)

• Fluorescent microscopy
• Fluorescence recovery after photobleaching

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Chromatin domain(1/2)

• Chromatin structure

• Domains on chromatin
• LCR locus control region
• 2. MAR matrix attachment regions
• 3. delimiter or scs (specialized chromatin
  structures)
• - prevent effects from spreading beyound the
• functional unit

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Chromatin domain(2/2)

• Two types of heterochromatin
• Constitutive (???) heterochromatin
• Facultative (???) heterochromatin

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Heterochromatin(1/4)

• Constitutive heterochromatin
• permanent feature of all cells
• includes centromeric and telomeric DNA
• ExampleY chromosome

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Heterochromatin(2/4)

• Facultative heterochromatin
• Not a permanent feature
• Contain genes that are inactive

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Heterochromatin(3/4)

• Euchromatin
• Matrix-associated regions (MARs) or Scaffold
  attachment regions (SARs)

• Euchromatin and Heterochromatin
• ???????????(euchromatin)?????(heterochromatin)???
  
• ??????????,??????????????,????,
• ???????
• 2. ?????????????????????,??????????????,
• ????????????????????

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Heterochromatin(4/4)
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Structural domain and Functional domain

• Structural domains
• Functional domains

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Insulators(??)(1/3)

• The boundaries of functional domains are marked
  by sequences
• scs and scs

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Insulators(2/3)

• Two special properties
• Overcome the positional effect
• Gene no longer responds to its regulatory
  modules
• Su(Hw)

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Insulators(3/3)
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Functional domains contain locus control
regions(1/2)

• Locus control region (LCR)
• Discovered during a study of the human -globin
  genes

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Functional domains contain locus control
regions(2/2)

• -globin LCR has shown that it contains five
  separate DNase I hypersensitive sites

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Activating the genome

• Histone modifications determine chromatin
  structure
• Nucleosome remodeling influences the expression
  of individual genes

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Histone modifications influence chromatin
structure

• Acetylation
• Deacetylation
• Phosphorylation
• Ubiquitination
• Ubiquitin
• Methylation

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Acetylation

• Histone acetylation reduces the affinity of the
  histones for DNA
• Histone acetyltransferases (HATs)
• Tetrahymena(???)protein---p55
• Mammalian protein---p300/CBP

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Deacetylation

• Reversing the activating effects of histone
  acetylation
• Histone deacetylases (HDACs)
• mammalian Sin3 complex
• yeast Sir2

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Histone acetylation is catalyzed by histone
acetyltransferases (HATs)
and histone deacetylation is catalyzed by
histone deacetylases (denoted by HDs or HDACs). 
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Nucleosome remodeling

• Nucleosome remodeling
• Remodeling
• Sliding(cis-displacement)
• Transfer(trans-displacement )

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Genome silencing by DNA methylation

• DNA methylation can repress gene activity
  (Cytosine methylation)
• DNA methyltransferase
• The methylation pattern is not random
• in higher eukaryotes5 CG 3
• in plants5 CNG 3

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DNA Methylation

• DNA methylation is the addition of a methyl group
  (CH3) to the DNA's cytosine base.
• It may affect gene transcription through several
  different mechanisms .
• Therefore, DNA methylation plays an important
  role in cell differentiation during development

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Methylation

• Maintenance methylation
• the methylation pattern is maintained after
  cell division
• De novo methylation

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CpG island

• In humans40-50 of all genes are located close
  to CpG islands
• CpG island reflecting the expression pattern of
  the adjacent gene

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How methylation influences genome expression

• Methyl-CpG-binding proteins(MeCPs)

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Methylation is involved in imprinting and X
inactivation

• DNA methylation and genome silencing is provided
  by two intriguing phenomena
• genomic imprinting
• X inactivation

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Genomic imprinting

• Only one of a pair of genes, present on
  homologous chromosomes in a diploid nucleus, is
  expressed
• The second being silenced by methylation

???????????????????????????, ???????????(turn
on)??? ??????????,????????????????? ??????????????
,????????,??????? ??????????????????????????????
,??????????????
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Genomic imprinting
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?????????????DNA??????(methyl group),?????? ???(me
thylation),???????????turn off????, ??????????????
????????,?????????? ??????? ???????????????????,?
????????????? ??????????????????
(imprinting),????????? ????????(turn
off)????????????????? (???????????????? ?????),?
??????????????,????????,?????????????,??????????
?????, ???????
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?????????1. Prader-Willi syndrome (PWS,
??-????)??????????15?q arm??????????,?????? ?????
???,?????2. Angelman syndrome (AS,
?????)????????????15?q arm????????,?????? ???????
?,?????3. Fragile X syndrome (???X????)X???????
?????? (CGG) ???????????,??? ?????????????????????
,??????? ??????,?????X?????????,?X????????? ??????
,????????????X????????, ?????,?????????????,????
(??????)?
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X inactivation

• One of the X chromosomes in a female mammalian
  cell
• Condensed X chromosomes structure called the Barr
  body