Bart Mol Biol 2009 - | SIU School of Medicinebbartholomew/Bart Mol Biol 2009.pdf · 2009-08-27 ·...

Contact informationBlaine BartholomewOffice: Neckers Bldg., Rm. 211 Phone: 453-6437 Email: bbartholomew@siumed.edu

Class noteshttp://www.siumed.edu/~bbartholomew/HbTxn.html

Other referencesPrinciples of Biochemistry by Lehninger 4th edition, pp 273-284, 938-943, 1082-1085, 1102-1104.

And for more in depth information (A) Thompson & Thompson, Genetics in Medicine, 1991 (B) Gelehrter, Collins, & Ginsburg, Principles of Medical Genetics, Second ed., 1998 (C)Connor & Ferguson-Smith, Medical Genetics, Fifth ed., 1996, (D) Jorde, Carey, Bamshad, & White, Medical Genetics, 2nd ed., 1999

Developmental Expression of Human Globin Genes

from Gelehrter, Collins, & Ginsburg, Principles of Medical Genetics, Second Ed., p. 93, 1998

ζ2ε2 α2ε2ζ2γ2

α2γ2 α2β2 α2δ2

Genomic Organization

Single copy: most common type with one copy/haploid

Duplicated genes: 2 or more copiese.g. histones

Gene families: closely related genes with similar but nonidenticalfunction

e.g. beta globin family

Pseudogenes: nonfunctional copies of functional genes that arise by gene duplication

e.g. b globin family

Lodish, Fig. 10-3b

β Globin Gene Family

Functional Genes (expressed at different times during development)ε, Gγ, Aγ, δ, β

Nonfunctional Pseudogenesψβ2, ψβ1

Human Globin Genes and HemoglobinsProduced During Development

β β

αα

Heme Developmental Period

Embryonic Fet al Adult

Hb Gower 1ζ2 ε2

Hb Gower 2α2 ε2

Hb Fα2 γ2

HbA2α2 δ2mi nor

HbAα2 β2

Hb Port landζ2 γ2

3’5’Chromosome 11 β-like genes

ε Gγ Aγ ψβ δ β

5’Chromosome 16 3’ α-like genes

ζ ζψ ψα ψα α2 α1

HbA

adapted from Thompson & Thompson Genetics in Medicine, p. 249, 1991

LCR

Sickle cell diseases

• Sickle cell anemia– Caused by a single point mutation

• Sickle cell-hemoglobin C• Sickle cell beta-thalassemia

– Caused by mutations in the β globin gene locus

– Decreased or absent adult β globin synthesis

One other version of these mutations

• Individuals with defective adult β globingenes can be phenotypically normally if …

• There are compensatory mutations that cause expression of fetal β-like genes after birth (Gγ and Aγ globin).

• This condition is called HPFH or hereditary persistence of fetal hemoglobin

How is the expression of these genes regulated?The important underlying DNA

sequences and associated factors

Eukaryotic & Prokaryotic TranscriptionRNA polymerases

RNA PolymerasesA. E. coli RNA polymerase

1. core enzyme = ββ'(α)2 has catalytic activity but cannot recognize start site of transcription

~500,000 daltonsdimensions: 100 X 100 X 160 angstromsrequires Mg2+ for activityb' binds 2 Zn atoms

2. holoenzyme = core enzyme + sigma factor (s)carries out four functions:

(i) template binding(ii) RNA chain initiation(iii) chain elongation (iv) chain termination

RNA PolymerasesB. Eukaryotic RNA polymerases (RNAP)

1. 3 nuclear RNA polymerases a. RNAP I- transcribes rRNA genesb. RNAP II - transcribes mRNA genesc. RNAP III - transcribes tRNA, 5S rRNA, and

other small RNA genesd. have 10-17 different subunits, large

multisubunit complexes are functionally similar to E. coli RNA polymerase

e. cannot bind to their respective promoters alone, but requires transcription factor for promoter specific recruitment

RNA Polymerases

2. organelle specific RNA polymerasesmore prokaryotic-like

1. chloroplast2. mitochondria

RNA Polymerases

3. RNAP IIa. core subunits - have sequence similarity to the

core subunits ofE. coli core RNA polymerase or subunits of other eukaryotic RNA polymerases

b. shared or common subunitssame subunits found in RNAP III and II or in RNAP I

and RNAP IIc. unique subunits - no similar homologs found

anywhere else

How does RNA polymerase II find its correct binding site?

Promoter StructureB. mRNA genes transcribed by RNAP II

1. TATA box element - located between -30 and -20 bps2. Initiator region or In: centered on the start site of transcription3. DPE: downstream promoter element4. Response elements (RE)

a. upstream of the TATA boxb. many different kinds - help respond to signalsc. multiple RE present - synergy

How is transcription of particular genes get turned on in response to external stimuli such as stress

(heat, starvation, and so on), hormones and other small molecule effectors?

Promoter Structure

B. mRNA genes transcribed by RNAP II5. enhancers

a. can be located at great distances (>1000 bps) from start site of transcription either from the 5' or 3' end of gene

b. stimulates transcription (~100 times)c. orientation independent

Promoter Structure

B. mRNA genes transcribed by RNAP II5. enhancers

d. two models of how enhancers might work i. entry point of RNAP II by preventing

nucleosomes from binding or an altered DNA conformation that promotes RNAP II recognition

ii. transcription factors bound to enhancer will stimulate binding of RNAP II to promoter regions closer to the start site of transcription

Transcription Factors

General versus promoter specific transcription factors.

Factors that are required for all mRNA genes and others that are required for only a small

subset of genes

General Transcription Factors

Promoter Specific Transcription Factors

General Transcription Factors

Regulation of TranscriptionI. Basal vs. activated transcription for mRNA genes

A. General transcription factor (TF) vs. promoter-specific1. general TFs are required by all mRNA genes

a. an absolute requirementb. transcription can occur alone with these

factors and is by definition the basal level of transcription2. promoter-specific TFs are different for each gene3. the promoter-specific TFs are required for

maximal level of transcription or for activated transcription (induction)

β − globin gene transcription factors

Erythroid specific Transcription Factors

TAFII130, CBP/p300

346

basic leucinezipper

TGCTGA(C/G)TCANF-E2

Sp1, GATA-1,FOG, EKLF,

CBP/p300

GATA-likeC4 zinc

fingers

(A/T)GATA(A/GGATA-1

GATA-1, CBP/p300, TAFII130

Kruppel-like C2H2 zinc

finger

CACCCEKLF

INTERACTS WITH

CLASSMOTIFPROTEIN

Regulation of TranscriptionII. Question of Activation

A. diversity of response - combinatorial effect1. properties of response elements (RE)2. relatedness of RE and enhancers3. trans acting factors

induction: heat shock, heavy metals, viral infection, growth factors, steroids

4. greater multiplicity with combinatorial approachB. Master gene regulatory proteins

1. response elements shared2. example of homeodomains

What is the difference between basal and activated transcription?

What’s wrong with this picture?

DNA is packaged into compact structures inside the cell

Higher order structure – folding of chromatin fibers

Differing extents of compaction

Also this facilitates in DNA fitting inside the nucleus

β− Globin Gene Cluster:Chromatin Structure

Gaining Access to the DNA

Large multi-subunit protein complexes alter the state of

chromatin

What is chromatin remodeling?

The process of making DNA more or less accessible in the eukaryotic genome using

a series of specialized proteins

Accessibility is Key Regulatory Step

ATP-dependent chromatin remodeling machinesDifferent classes of chromatin

remodeling complexes

Different classes of ATP-dependent chromatin remodeling complexes

A. SWI/SNF or SWI-SNF like complexes– required for activation of a small subset of

genes– found in humans, flies, and yeast– at least two different forms of the complex in

yeast or humans– in yeast it has 11 or 17 subunits– total complex size is 1.5 -2 Megadaltons

SWI/SNF or SWI/SNF-like Complexes Are Found in All Eukaryotes

Activities associated with ATP-dependent chromatin remodeling• Translate nucleosomes along DNA

– Change translational position (general)– Nucleosome spacing (ISWI)

• Displace or dissemble nucleosomes (SWI/SNF and RSC)

– Octamer transfer– Loss of H2A/H2B dimer

• Histone exchange– Example of H2A exchanged for H2AZ by SWR1

Bulge Diffusion Model for chromatin remodeling

• Initiates from outside the Nucleosome• Initially breaks contacts at edge of Nucleosome•These two point are also true for other model(s)

DNA can be cut by nucleases (LCR)

Site 3 has SWI/SNF bound at this region

Chromatin Remodeling • ATP-dependent chromatin remodeling

– Moves nucleosomes along DNA– Involved in displacing nucleosomes from DNA– Exchanges parts of the nucleosome

• Covalent modification of histone proteins– ACETYLATION– METHYLATION– PHOSPHORYLATION– UBIQUITINATION– Poly ADP RIBOSYLATION– SUMOLYATION

Sites of Histone Modification

Histone acetyltransferases (HATs)

A. cytoplasmic HATs (Type B)i. acetylate newly synthesized H3 and H4 histones

before depositing on to DNAii. after binding to DNA the acetyl groups are

removed

B. nuclear HATs (Type A)i. have been found to be required for gene

activationii. part of TFIID has HAT activity (TAFII250)iii. these HAT proteins are usually large multi-

subunit complexes

Mapping sites of histone modifications

• Have developed a technique called ChIP(Chromatin Immunoprecipitation)

• Identify the genomic location where different histone modifications reside

• Is dependent on antibodies that recognize the particular modification

• Can be used to examine the entire genome

Pattern of histone acetylation on the β globin gene

Chromatin

Euchromatin - dispersed (acetylated) chromatin, gene rich regions of the genome

Heterochromatin - chromatin is condensed (deacetylated), gene poor regions of the genome

human genome 3 x 109 bp with an estimated 20-25,000 genes only 2- 4% of the DNA codes for proteins

Histone acetylation generally associated with gene activation

Summary of regulatory elements

What is an insulator element (which is similar to what is also called a boundary element)?

Insulators: Two models

Nuclear Matrix or Scaffold

DNA Loops/ Domains

Chromosome Territories

Individual chromosomes occupy distinct regions in the nucleus

Chromosome Territories

• Gene rich chromosomal regions– Tend to be oriented toward the nuclear

interior• Gene poor chromosomal regions

– Tend to be associated with the with nuclear periphery

• Dynamic reorganization