Gene Regulation 5 - Regulation of eukaryotic gene expression - Transcriptional regulation

Question 1

Learning Outcomes

Answer 1

Students will be able to
➢ explain the terms housekeeping, constitutive, facultative, induced and repressed genes and how this
relates to gene expression in different cell types.
➢ list the type of genes transcribed by RNA polymerases in prokaryotic and eukaryotic cells.
➢ compare the general structure of prokaryotic and eukaryotic protein coding genes.
➢ describe different promoter elements of eukaryotic, RNA polymerase II transcribed genes.
➢ describe different types of transcription factors and how they regulate the expression of RNA
polymerase II transcribed genes.
➢ explain how epigenetic modifications and chromatin remodeling contribute to the regulation of gene
expression and development.

Question 2

Regulation of gene expression in eukaryotes

Answer 2

• Control of gene expression in eukaryotes occurs at several
  levels:
  1) Packing or unpacking DNA
  2) Transcription
  3) mRNA processing
  4) mRNA export
  5) Translation
  6) Post-translation protein modification
  7) Protein degradation

Question 3

Genes are expressed at different levels

Answer 3

• Only subsets of genes are expressed in a cell type ➔ expression levels of genes varies from one cell type to another.
  – depends on cell type and internal or external conditions of the cell, e.g. changes in a cell’s environments or cell cycle
  stage.
• Expression of all genes requires regulatory elements. These are DNA sequence motifs in promoters (cis-acting elements)
  and transcription factors (trans-acting factors). These vary between genes.

Answer 4

Question 5

Housekeeping Genes are expressed in all cell types

Answer 5

• Some genes are expressed in all cells: housekeeping genes (here gene B).
  – can be constitutive expressed genes = transcribed continually.
  – can be expressed at a relatively constant levels or have varying expression levels.
  – required for the maintenance of basic cellular functions, vital for survival.
• Examples: RNA polymerase, DNA repair enzymes, structural proteins of
  chromosomes, enzymes of basic metabolic processes.

Answer 6

Question 7

Facultative Genes are expressed when the gene product is needed

Answer 7

• A specific cell only expresses a subset of the genes in its genome.
• Facultative genes ➔ only expressed when required (here gene A and C)
  – Inducible and repressible genes ➔ expression is responsive to changing internal or external conditions, they may
  respond to environmental change or they may be dependent on the cell cycle.

Answer 8

Question 9

Eukaryotes use three different RNA polymerases
for different gene types

Question 10

Prokaryotes:
* have only one RNA polymerase, transcribes all
prokaryotic genes
* Can initiate transcription on its own
– The RNA polymerase sigma subunit positions the
polymerase at the promoter

Answer 10

Question 10

Eukaryotic protein encoding genes are more complex than
Prokaryotic genes

Question 11

Eukaryotes:
* RNA polymerase I: ribosomal RNA genes: 5.8S rRNA, 18S rRNA & 28S rRNA genes
* RNA polymerase II: all protein-coding genes ➔ mRNAs
* small nucleolar RNA genes: guide chemical modifications of RNAs such as
tRNAs; some small nuclear RNA genes: help in mRNA splicing; microRNA
genes: involved in the regulation of gene expression
* RNA polymerase III: tRNA genes, 5S rRNA gene, some snRNA genes, other small
RNA genes
* Each eukaryotic RNA polymerase requires different promoter elements and
different transcription factors

Answer 11

Question 12

Prokaryotic genes:
▪ Often polycistronic = encode for
more than one protein
▪ Organised in operons
▪ few shared, simple regulatory
elements (promoter, operator)

Answer 12

Question 13

Eukaryotic genes:
▪ Usually monocistronic =
encode for one protein
▪ Most have introns
▪ Very complex promoters:
reflect complexity of
organism with many cell
types, many functions

Answer 13

Question 14

Eukaryotic promoter of RNA polymerase II transcribed genes
are more complex than prokaryotic promoters

Answer 14

Question 15

Cell specific Regulation of transcription requires a large variety of
Transcription Factors (TFs)

Answer 15

Two main categories of eukaryotic transcription factors:
* General TFs: bind to core promoter elements, help RNA polymerase to initiate transcription
* Cell or tissue specific TFs: transcription regulators that bind to promoter proximal elements or to distal
promoter elements, DNA sequence specific, modulate expression levels in response to signals.

Question 16

Eukaryotic promoter of RNA polymerase II transcribed genes
are more complex than prokaryotic promoters

Answer 16

• Core Promoter: determine the transcription start site & direct binding of RNA pol II. Contain TATA box in rapidly
  transcribed genes
• Transcription control regions: mediate cell type specific expression and activate transcription. Short (6-10 bp) DNA
  sequence elements that bind activating transcription factor proteins.
  – Promoter proximal elements: close to transcriptional start site (within 200 bp)
  – Distal Promoter elements: Enhancer or Repressor Elements: far from transcriptional start site (up to 50,000 bp)

Question 17

General Transcription Factors position
RNA polymerase II at the Core Promoter

Answer 17

• General Transcription Factors have a similar
  role in eukaryotic transcription as the sigma
  factor has in bacterial transcription.
• They assemble sequentially at the core
  promoter of protein encoding genes:
  – The TBP (TATA Binding Protein), a subunit
  of TFIID binds to the TATA box in the core
  promoter
  – TFIIB, TFIIE and TFIIH assemble then
  sequentially
  – TFIIF positions the RNA polymerase II at the
  transcription start site
• Preinitiation complex
  – TFIIH uses energy (ATP) to pull apart the
  DNA double helix, exposing the template
  strand ➔ allows RNA pol II to begin
  transcription

Answer 18

Answer 19

Question 20

Cell or Tissue specific Transcription factors:
DNA binding proteins that can form protein dimers

Answer 20

➢ DNA binding proteins
➢ Often bind as dimers = two TF proteins
➢ TF DNA binding domain ➔ amino acid
sequence that interacts with DNA
➢binds to TF specific DNA sequence motifs in
promoters = gene regulatory sequences
➢ TF activation or repression domain ➔ amino
acid sequence that regulates gene expression

Answer 21

Question 22

Combinatorial control of gene expression

Answer 22

➢ Many transcription factors are active at any one
time in a cell
➢ They may interact with:
➢ RNA polymerase
➢ each other - homodimer
➢ other TFs - heterodimer
➢ TFs can act as activators, co-activators or
repressors of transcription
➢ Repressors of transcription interfere with the
binding of a transcription factor to the DNA
elements
➢ The combination of TFs bound to regulatory
DNA elements determines outcome =
combinatorial control

Answer 23

Question 24

The combination of transcriptional regulators can generate many cell types during development

Answer 24

* After each cell division, a new type of transcriptional regulator is made. * This leads to a variety of combinations of transcriptional regulators over several cell generations. * Each combination will lead to the expression of a different gene subset. * This leads to many different cell types

Answer 25

Question 26

Some transcription factors can be activated

Answer 26

Answer 27

Example: * Gene 1, 2 and 3 have TFs bound to their respective promoter elements (regulatory DNA sequences) * These TFs are not sufficient to activate transcription efficiently – Low gene expression * The presence of cortisol activates the cortisol receptor forming a cortisol receptor complex * Activated cortisol receptor complex binds to the cortisol receptor binding sequence. – This sequence is found in promoters of gene 1, 2 and 3 – activates efficient transcription, genes 1, 2 and 3 are expressed at higher levels

Question 28

The regulatory effect of many transcription factors is mediated by the Mediator Complex

Answer 28

* The general transcription factors assembled at the core promoter are the same for all RNA polymerase II transcribed genes. * Different gene promoters have different combinations of binding sites for different transcriptional regulators. * A typical eukaryotic gene is controlled by many transcription regulators * The regulator binding sites can be far apart. * A multiprotein complex named the mediator complex brings the activities of transcriptional regulators together so that they can impact on transcription by helping to assemble: * RNA polymerase II * general transcription factors * chromatin remodelling complexes * histone modifying enzymes R G C H

Answer 29

Answer 30

Question 30

Eukaryotic transcriptional regulators can recruit chromatin modifying proteins and histone modifying proteins

Answer 30

Last lecture: * Nucleosomes can physically prevent accessibility of proteins (TFs, RNA pol etc) to DNA * Chromatin remodelling complexes can change chromatin structure. * Histone modifying proteins can change chromatin structure. ➔ they influence the accessibility of DNA to transcription factors, e.g. exposure of the TATA box. * Activating transcription regulators can recruit chromatin modifying proteins via the mediator complex that open up chromatin. * Repressing transcription regulators can recruit chromatin modifying proteins via the mediator complex that close chromatin.

Question 31

Epigenetic modifications contribute to gene regulation: histone modifications

Answer 31

* Last lecture: histone modifications can impact on gene expression. * During chromosome replication, histones are distributed randomly to daughter DNAs. – Daughter cells have half of the parental histones and half of the histones are newly synthesised. * Parental pattern of histone modifications is re-established by histone modifying enzymes ➔ Genetic modifications that impact gene activity without changing the DNA sequence: Epigenetics

Answer 32

Answer 32

Question 33

Epigenetic modifications contribute to gene regulation: DNA methylation

Answer 33

▪ DNA methylation = addition of methyl groups to DNA by DNA methyltransferases: DNMT ▪ DNA CpG (= C phosphate G) methylation is symmetrical = occurs on both DNA strands ▪ DNA methylation changes activity of a DNA segments without changing the sequence = epigenetic modification ▪ DNA methylation in a gene promoter typically represses transcription of the gene.

Question 34

DNA methylation is both dynamic and essential for normal development

Answer 34

Question 34

DNA methylation patterns can be inherited by daughter cells

Answer 34

Question 35

Epigenetics is influenced by “Lifestyle”

Answer 35

Comparative genomic hybridization for methylated DNA: Green: hypermethylation, red: hypomethylation, yellow: equal amounts of hyper and hypomethylation. Chromosome 17 as an example * 3-year-old twins have a very similar distribution of DNA methylation. * 50-year-old twins Shave significant DNA methylation changes.

Question 36

Epigenetics can cause monozygotic (MZ) twin discordance in schizophrenia

Answer 36

Epigenetic model of monozygotic (MZ) twin discordance in schizophrenia. Red circles represent methylated cytosines. Phenotypic disease differences in MZ twins result from their epigenetic differences. These can be caused by gene hypomethylation (as shown) or gene hypermethylation (not shown).

Answer 36

Question 37

Hyper-methylation of the FMR1 gene promoter causes Fragile X Syndrome

Answer 37

* FMR1 = fragile X mental retardation 1 gene * FMR1 encodes a protein, most commonly found in the brain, that is essential for normal cognitive development and female reproductive function The FMR1 promoter mutation: * Expansion of a CGG triplet repeat within the FMR1 gene: normal 5 to 40 copies, expansion > 200 copies. * Hyper methylation of the promoter due to increase in CGG repeats causes repression of FMR1 gene. * This causes a lack of FMR protein ➔ disrupts nervous system functions and leads to the signs and symptoms of fragile X syndrome

Answer 37

Question 37

Mis-methylation can have detrimental effects on development

Answer 37

Fragile X syndrome – An example of mis-methylation leading to detrimental effects – Common form of a genetic or inherited condition: 1 in 1500 to 4000 males, 1 in 2500 to 8000 females. – A range of developmental problems: * learning disabilities and cognitive impairment, * Autism and Parkinsonism * premature ovarian failure. – Cytologically fragile site on X-chromosome causing breaks in vitro.

Question 38

Epigenetic modifications differ between normal cells and tumour cells in humans

Answer 38

Normal cells: ▪ CpG islands preceding gene promoters are generally unmethylated = transcriptionally active Cancer cells: ▪ CpG islands preceding tumour suppressor gene promoters are often hypermethylated = inactive ▪ CpG methylation of oncogene promoter regions repeat sequences is often decreased = active Manipulation of epigenetic alterations may be useful for cancer prevention, detection, and therapy

Answer 38

Question 39

Can you …

Answer 39

➢ … classify genes according to their regulation and to when and where they are expressed? ➢ … compare RNA polymerases from pro- and eukaryotes in relation to the genes they transcribe? ➢ … compare the structure of prokaryotic and eukaryotic protein coding genes, including the differences in their promoters? ➢ … explain the need for general and cell/tissue specific transcription factors in eukaryotes and what their role is during transcription initiation? ➢ … explain how the combination of transcriptional regulators impacts on the development of many cell types? ➢ … explain how the activity of many transcription factors is mediated to achieve regulation gene expression? ➢ … using examples, explain the principle of transcription factor activation? ➢ … integrate the action of transcription factors with chromatin modifications and epigenetic control? ➢ ... explain how DNA methylation work in the context of epigenetic control of gene expression and how it changes during development? ➢ … relate diseases to malfunctions of epigenetic control?