Friday, November 8th Lecture Notes
In both bacteria and eukaryotes, transcription is primarily regulated by transcription factor proteins that bind to DNA sequences associated with any given gene
But in eukaryotes, transcription is also regulated by the structure of the chromatin surrounding the gene
Chromatin structure is controlled at two levels:
Modification of the histones associated with the DNA
Methylation of the DNA itself
In a eukaryotic chromosome, genomic DNA is thought to be packaged into a series of large loops attached to a proteinaceous scaffold
At any given time, most of the DNA in a eukaryotic cell is wound around nucleosomes
A core promoter or other essential regulatory sequence can be blocked if it is pressed against the histones
But cells express such genes with the aid of chromatin remodeling, a process in which nucleosomes are either moved or disassembled to expose the blocked sequences
Pioneer TFs activate chromatin remodeling
Saccharomyces cerevisiae
A single-celled eukaryote widely used to study cell and molecular biology
Yeast regulate expression of metabolic enzymes depending upon the nutrients available in its environment
When the sugar galactose is present, the transcription factor Gal4 activates transcription of a set of genes required for galactose metabolism
The Gal4 homodimer binds to a DNA sequence known as UAS
Gal4’s consensus binding sequence is called USA (upstream activating sequence)
The cis-regulatory region of the Gal1 gene contains 4 UAS sequences, together referred to as the UASg enhancer
Gal4 must bind one or more of these sites in order to activate Gal1 transcription
Eukaryotic enhancers often have multiple binding sites for the same transcription factor; the more molecules of Gal4 that are bound, the higher the rate of Gal1 transcription
But yeast is a eukaryote, and its DNA is wrapped into nucleosomes; how does Gal4 gain access to the UAS binding sites?
The UASg enhancer also contains binding sites for the RSC chromatin remodeling complex; RSC insures that the enhancer DNA is associated with partially unwound nucleosome which leaves the Gal4 binding sites accessible
Once Gal4 is bound to the UASg enhancer it uses its activation domain to recruit a second chromatin remodeling complex called SWI/SNF
SWI/SNF displaces a nucleosome that covers the core promoter and +1 start site of target gene Gal1
The Gal4 transcription factor plays multiple roles in regulating gene expression:
When Gal4 binds to the UAS sequences, it induces chromatin remodeling to expose the Gal1 promoter
Gal4 also plays a role in recruiting the transcriptional machinery to the exposed promoter
Using its activation domain, Gal4 protein bound to the UAS enhancer interacts directly with the general transcription factor TFIID and the mediator complex—attracting them to the Gal1 promoter
This leads indirectly to the recruitment of RNA Pol II, and the activation of Gal1 transcription
Chromatin can also be remodeled by chemical modifications of the histones that form the nucleosome core
Chromatin structure can be modified by the acetylation, phosphorylation, and/or methylation of amino acid R groups on the N-terminal tail domains of the core histones
Dr. Marie Maynard Daly
Identified the amino acid sequence of histones and discovered that regions were lysine rich
Also, groundbreaking work on nucleic acids, protein synthesis, cholesterol, hypertension and atherosclerosis, and creatine and muscle cells
Chemical modifications of histones influences the rate of gene transcription
Acetylation and phosphorylation of histone tails promotes the transcription of the neighboring gene(s)
Methylation of histone tails generally represses the transcription of the neighboring gene(s); however, the effect of histone methylation can vary depending upon exactly which amino acid is methylated
Histone acetylation is the most common form of modification
Histone acetyltransferase (HATs) can covalently bond an acetyl group to a lysine R group
Acetyl groups can be removed from the R group by histone deacetylases (HDACs)
Acetylation and deacetylation change the net electrical charge of the histone protein subunit
Chromatin structure has important implications for the transcription potential of the DNA
Heterochromatin has little or no transcription
Euchromatin has a high level of transcription
The N-terminal histone tails are required for formation of the 30 nm fiber, and studies suggest that the positively charged tail of histone H4 is a key player
Acetylation of H4 by HAT negates its positive charge, causing disassembly of the 30 nm fiber and locally increasing the rate of transcription
Deacetylation by an HDAC can reverse this process
Reduce the rate of transcription
Once histone tails are acetylated, they can recruit other proteins that contain bromodomain motif
This includes nucleosome remodeling proteins and additional histone acetyltransferases
These proteins work together to increase the accessibility of DNA for transcription
Methylated histone tails can recruit proteins that contain chromodomain motifs
Many of these chromodomain proteins help to silence transcription of the surrounding DNA
DNA methylation has different functions in different organisms:
E. coli uses DAM methylase to distinguish new and parent DNA strands during base mismatch repair
In addition, methylation protects bacterial DNA from the cell’s own restriction enzymes
DNA methylation does not play either of those roles in eukaryotes; the primary role of DNA methylation in animals is to regulate chromatin structure and silence gene transcription
In mammals, the enzyme DNA methyltransferase can add a methyl group to carbon 5 of the cytosine ring
Methyl-C still base pairs with G, so there is effectively no alteration in the base pair sequence of the DNA
The main target for DNA methylation is the sequence CpG
To mark the gene for silencing, a methyltransferase methylates both strands of the CpG
In an adult human approximately 70% of CpGs are methylated
The majority of CpG sites are clustered in the cis-regulatory regions of protein-coding genes; these clusters are called CpG islands
While methylation of a single CpG has little or no effect on the gene, methylation of multiple sites within a CpG island can result in gene silencing
Experimentally removing CpG methylation activates the transcription of silenced genes
In the KM20 tissue culture cell line, the neurotensin gene is transcriptionally silent
These cells show heavy CpG methylation of the NT promoter
CpG methylation can be blocked with the drug 5-azacytidine
Applying this drug to KM20 cells eliminates DNA methylation and reactivates transcription of the neurotensin gene
DNA methylation and chromatin remodeling reinforce one another in regulating chromatin structure
Epigenetic gene regulation
In many cases gene silencing is faithfully transmitted over multiple generations of cell division
In some cases, it can be inherited from an organism to its offspring
Epigenetic = information is being passed along without any modification in the DNA sequence of the gene(s)
Persist states of gene regulation
When a cell divides, its pattern of DNA methylation is reliably passed along to both daughter cells:
DNA replication of a methylated CpG produces two hemi methylated CpGs
An enzyme called maintenance methyltransferase follows the replication fork, restoring the fully methylated CpGs in the same gene(s) of both daughter cells
Epigenetic gene regulation plays an important role in the sex chromosomes of humans and other placental mammals
The human X chromosome contains approximately 2,000 protein coding genes
If men and women expressed these genes at the same level, women would synthesize twice as much of all 2,000 proteins.
This genetic imbalance would be lethal
To compensate, XX females silence the transcription of 1 of the 2 X chromosomes in each cell
Recent studies indicate that X inactivation is not complete; the inactivated X actually retains about 5% of the transcriptional activity of the active X
Inactivation of a female X chromosome involves hypermethylation of its DNA and hypoacetylation of its histones
The inactivated chromosome completely condenses into heterochromatin, a structure called a Barr body
Inactivation occurs in XX females at a time when the embryo is composed of 32-64 cells
Inactivation is random; the maternal X or the paternal X can be inactivated in any given cell
Once an embryonic cell has inactivated one X chromosome, that chromosome remains inactive in subsequent cell divisions, an example of epigenetic gene regulation
Because X chromosome inactivation occurs randomly in different cells, the body of an XX female mammal is a patchy mosaic of tissues expressing the maternal or paternal X chromosome
For example, tortoiseshell and calico cats are XX females heterozygous for an X-linked gene that has distinct black and orange alleles
Anhidrotic Ectodermal Dysplasia
A human disease condition caused by a loss-of-function allele of an x-linked gene essential for sweat gland and hair development
Although X chromosome inactivation is inherited from cell-to-cell as the body grows, it is not inherited from mother to child
Why not?
If it were, one half of the XY offspring would inherit the mother’s inactive X chromosome and that would be lethal
To minimize the inheritance of gene silencing from parent to child, there is a global demethylation of genomic DNA within the germ cells
DNA methylation is not erased in somatic cells, but they do not contribute DNA to the next generation
In this way, the inactivated X chromosome gets reactivated before it is passed along to children
Mini Study Guide
Chromatin structure is regulated by modifying histones and by modifying the DNA itself
Chromatin remodeling involves moving or removing histones
Give an example of a gene that is regulated by chromatin remodeling, and explain how that chromatin remodeling takes place
Chromatin modification involves acetylation, phosphorylation, or methylation of histone tails
Know how each of those modifications are achieved and what they do to chromatin and to gene expression
Know how and where DNA is methylated
Explain how and why X inactivation occurs
I-Clicker Questions
What type of protein can bind to DNA that is wound around nucleosomes and compacted?
Pioneer transcription factor
Gal4 transcription factor binds to the UAD sequences in an enhancer that regulates the GAL1 gene. Why can’t it bind without assistance
The UAS binding sites are normally obstructed by nucleosomes
Nuclear remodeling complexes do all of the following except:
Recruit transcription factors
Which of the following are nucleosome remodeling complexes?
RSC
SWI/SNF
The Gal4 transcription factor regulates Gal1 transcription through all of the following mechanisms except:
Interacts directly with RNA Pol II to initiate transcription
To maintain tightly compacted chromatin, the histone tails should be:
Positively charged
Which enzyme adds an acetyl group to negate the positive charge on lysine’s?
Histone acetyl transferase
Methylation of lysines in the histone tail does not remove the positive charge. What is the usual effect on transcription of nearby genes?
Repressed expression
Which proteins bind to bromodomains, and which ones bind to chromodomains?
Bromodomains bind acetylated lysines, and chromodomains bind methylated lysines
Where is eukaryotic DNA methylation found?
CpG islands
Where are these CpG islands located?
In cis-regulatory regions
What do we mean when we talk about epigenetics?
Chromatin state that is maintained as cells divide
When cells divide, which enzyme helps propagate the epigenetic state of each region of DNA?
Maintenance methyltransferase
How is X inactivation achieved?
Increased methylation of its DNA and reduced acetylation of its histones
George has Klinefelter syndrome…
George inherited an X chromosome from his mother, and both an X and a Y chromosome from his father
Which of the following is not inherited from parent to progeny in eukaryotes?
Methylation of the DNA