Ch. 17 - Regulation of Transcription in Eukaryotes Flashcards
Transcriptional regulation in eukaryotes
Transcriptional regulation is much more complex in eukaryotes than in prokaryotes.
Plants: 1400 Transcription factors (TFs)
Human: 1600 TFs
Why do we have so many TFs?
Many different types of cells, require different genes expressed.
- Expression may require many activators
- Enhancers both up- and downstream
- TFs often have to be transported into the nucleus
Controlling gene expression in eukaryotes is complicated by the high number of genes and transcription factors, packing of chromosomes, and the segregation of the chromosomes in the nucleus.
Transcription factors (TFs)
TFs recognize and bind to 4-8 nucleotides. They have two independent domains:
DNA binding domain: which recognizes a specific sequence in the DNA (normally 4-8 bases)
Activator domain: that interacts with the transcription machinery/transcription factors in the mediator complex.
One TF can regulate more than one gene.
Specific TFs typically share four properties:
- They respond to a stimulus which signals that one or more genes should be expressed
- Unlike most proteins, TFs are capable of entering the nucleus where the genes reside. Many steroid receptors are TFs
- They recognize and bind to a specific sequence on the DNA
- They also make contact with the transcription apparatus, either directly or indirectly.
The mediator complex
General TFs recognize and bind to the promoter. Activators are needed to initiate transcription, and these interact with the general TFs. In addition a large protein complex called the mediator is required (in humans it contains 26 subunits). It provides interaction with RNA polymerase II, and act as a binding interface for other transcription activators (or repressors), in particular enhancer proteins.
The mediator complex works as a Scaffold protein that helps activators to bind to the transcription complex, and assembles the pre-elongation complex, which initiate transcription. It enhances RNA polymerase II recruitment and stabilizes tha transcription complex at the promoter. It transmits multiple signals from TFs to the RNA polymerase to regulate transcription of genes.
Enhancers and insulators
Enhancers: increases expression by binding activators. Can be located upstream and downstream of, and thousands of base pairs away from the gene they control. Active in both forward and backward orientation. Enhancers act by looping the DNA so that the activator proteins bound to the enhancers can make contact with the transcription apparatus via the mediator complex.
Insulators: Special sequences that divide chromosomes into regulatory neighborhoods. Insulators bind clusters of specific zinc-finger proteins, IBP (insulator binding proteins), such as CTCF. These limit the activity of enhancers and prevent them from acting outside a defined chromosomal region, by isolating them from the rest of the DNA in a loop. Thus, preventing them from interfering with the wrong genes. Insulators can be inactivated by methylating their GC sequences.
There are 15 000 binding sites for CTCF in the human genome.
Matrix attachment region (MAR)
The nuclear matrix, a protein network, act as a scaffold for DNA. The DNA is attached to the proteins of the matrix by sites called MARs. This results in looping of the DNA with sizes of 60-100 kbp (0.2-1 kbp in AT-rich regions). MARs are often found near insulators sites.
Negative regulation: blocking activator binding site
In eukaryotes, negative regulation is often accomplished by interfering with activators or the mediator complex, rather than interacting with the DNA it self, often by blocking the binding site of an activator. The CAAT-binding factor (CTF) can be prevented from binding the CAAT box (small DNA motif) by the CAAT-displacement protein (CDP).
In eukaryotes, positive regulation is more common than negative. Opposite for prokaryotes.
Negative regulation: heterdimerization
Basic helix-loop-helix proteins (bHLH) has one helix containing basic amino acid residues that facilitated DNA binding. bHLH proteins bind DNA as dimers, but only bind DNA if both proteins contain the basic helix region.
The TF MyoD can form heterodimers with the bHLH protein E12 or the ID protein
- E12: bHLH protein, which binds DNA
- ID: protein lacking the bHLH, won’t bind DNA
Chromatin
Heterochromatin: condensed, densely-packaged DNA, cannot be transcribed because it cannot be accessed by RNA polymerase
Euchromatin: DNA accessible for RNA polymerase, can be transcribed
Acetylation of histones: HATs and HDACs
The histones of the nucleosome core (H2A, H2B, H3, and H4) have tails of about 20 amino acids at their N-terminal ends that faces outwards from the core, that can be acetylated at their lysine residues (most often H3 and H4). The degree of acetylation affects the state of nucleosome aggregation and therefore gene expression. Acetylated histones form less condensed chromatin, making the DNA more accessible for transcription.
Histone acetyl transferases (HATs) add acetyl groups and histone deacetylases (HDACs) remove them. Several proteins previously known as co-activators are actually HATs, and several co-repressors are actually HDACs. HATs and HDACs, and Co-activators and -repressors in general, do not bind DNA itself, but TFs that have already bound to the DNA.
Chromatin remodeling complexes
In addition to disaggregating the nucleosomes, a modification of the nucleosome architecture is needed to allow access of condensed genomic DNA. This is carried out by chromatin remodeling complexes, and includes sliding of nucleosomes (ISWI or Swi/Snf) or rearrangement by merging of nucleosomes (Swi/Snf).
Swi/Snf is a larger complex consisting of 8-12 proteins that bind strongly to DNA. These complexes can both slide nucleosomes along the DNA to reveal promoters, and remodel nucleosomes by merging two nucleosomes into one with a looser structure.
ISWI contains 2-6 polypeptides and can only slide the nucleosomes along the DNA as it binds to nucleosomes rather than DNA.
Binding of the chromatin remodeling complexes by TFs, targets them to the stretch if DNA that needs opening up. It is about 200 times easier to access the promoter if it is not in a nucleosome.
Activation of a typical eukaryotic gene
The precise order in which transcription factors, histone acetyl transferases, and chromatin remodeling complexes associate with the promoter appears to be promoter dependent. A generalized sequence of events for activating a typical eukaryotic gene is as follows:
- A TF binds to the DNA
- A HAT binds to the TF
- The HAT acetylated the histones in the nearby region, and the association of the. nucleosomes is loosened
- The chromatin remodeling complex slides and/or rearranges the nucleosomes, allowing access to the DNA
- Further TFs bind
- RNA polymerase binds to the DNA
- Initiation requires a positive signal to be transmitted via the mediator complex from one or more specific TFs
The Histone code: histone modifications
The histone code: the wide range of possible histone modifications. There are several chemical modifications of histone proteins. These modify chromatin structure and affect the binding of a variety of TFs and other proteins to DNA, thus affecting the gene expression. The most common modifications are:
Acetylation: opens up the chromatin structure, whereas deacteylation promotes aggregation of chromatin. The acetyl groups come from acetyl-CoA (link to metabolism).
Methylation: of lysine residues at position 4 and 36 in histone H3 promotes transcriptional activation, while methylation of lysine residues at positions 9 and 27 in histone H3 promotes repression of transcription.
Phosphorylation: may activate or repress transcription depending on location.
Ubiquitination: may activate or repress transcription depending on location.
Rare modifications including ADP-ribosylation, sumoylation, and biotinylation.
Large protein complexes called polycomb group (PcG) proteins are transcriptional repressors, as they methylate the H3K27 and ubiquitinate H2A.
Methylation of DNA
In vertebrates, methylation of cytosine is a powerful mechanism to pass on gene expression patterns to progeny (offspring) cells. The methylation of cytosines means that the pattern of DNA methylation can be inherited by the daughter DNA strands. Higher eukaryotes methylate up to 10% (plants 30%) of their cytosines. Methylation occurs at specific CG pairs in the DNA double helix.
Methylation of genes in eukaryotes often silence gene transcription (inhibitory). The methylation is often associated with GC-islands. Housekeeping genes often un-methylated GC-islands, as they need to be expressed at all times.
DNA methylases
There are two types of DNA methylases:
De novo methylases: add a methyl group to an un-methylated CpG site in a GC-island.
Maintenance DNA methylase: add a new methyl group to the opposite strand of the hemimethylated sites during DNA replication.
DNA demethylases remove methyl groups.
Gene silencing through DNA methylation
Silencing involves the covalent modification of both DNA and histones. It can involve a single gene or affect a large chromosomal region. The methyl groups project into the major groove of the DNA and can hinder the binding of TFs.
There are also specific proteins that recognize and binds to methylated CpG-sites. MethylCpG-binding proteins are recognized by other proteins such as histone deacetylases (HDACs), which by removing acetyl groups from histones may promote chromatin condensation and gene silencing. Genes are silenced by methylation of the DNA, followed by removal of acetyl groups from the histones.
