Lecture 12: Transcription Regulation II

Regulation of Transcription Side notes: Eukaryotic TFs recruit other regulatory complexes Eukaryotes typically regulate transcription via DNA binding proteins that recruit co-activators or co-repressors One example is ELK1, which recruits mediator, which is generally needed for RNA poi transcription Mitogens are extracellular signals that promote entry into mitosis In the absence of mitogens, ELK1 binds to serum response factor (SRF) but doesn't activate transcription Mitogen binding at the cell surface activates kinases, which phosphorylate ELK1. The phosphorylated protein recruits mediator, promoting transcription Gal4 recruits RNA polymerase through a co-activator complex Gal4 regulates genes that are responsible for galactose metabolism Gal4 activates transcription by binding to the UAS(G) sequence Gal4 activity is regulated by Gal80 and Gal3, which respond to galactose in the cell In the absence of galactose, Gal80 binds to Gal4, preventing transcription When galactose is present, Gal3 binds to Gal80, preventing Gal80 binding to Gal4, and allowing Gal4 to recruit SAGA and mediator, which activates transcription Hsf regulates transcriptional elongation & promoter proximal pausing Transcription elongation can be regulated by proteins alone, as with transcription of Drosophila hsp70. Hsp70 is involved in protecting the cell during abnormally high temperatures (heat shock) RNA polymerase sometimes pauses a er transcribing the first few dozen nucleotides (proximal pausing). This is used to prime hsp70 for rapid transcription in response to heat shock Without heat shock, GAGA factor binds upstream of hsp70 and recruits NURF, which keeps the promoter free of nucleosomes and allows polymerase binding Transcription starts, but at 25 degrees Celsius it pauses as the polymerase complex is not phosphorylated enough With a sudden rise in temperature, heat shock factor (hsf) trimerizes and binds to heat shock elements (HSEs) Hsf then recruits mediator to the promoter, as well as pausing complexes and a kinase, which phosphorylates the CTD and allows transcription to resume Yeast cell type is determined by four transcriptional regulators Combinatorial control is the response of a gene to multiple signals and regulatory proteins Yeast cell type is controlled by four transcriptional regulators A and alpha cells are haploid A/alpha cells are diploid and result from mating of a and alpha cells A and alpha can mate, but not a and a or alpha and alpha (i.e. Cells must be of di erent mating type) A/alpha cells cannot mate, but can undergo meiosis when starved a, alpha, and a/alpha lens have distinctive gene expression pa erns, regulated by four TFs: a1, alpha1, alpha2, MCM1 These regulators are encoded at the MAT locus on chromosome III The MAT loci are di erent in di erent cell types: a cells encodes a1, alpha cells encode alpha1 and alpha2 MCM1 activates transcription of a-specific genes. a-specific genes are not transcribed in alpha cells Similarly, alpha1 activates transcription of alpha-specific genes in alpha cells, Regulation of Transcription Side notes: while alpha2 represses a-specific gene transcription In a/alpha diploid cells, a1 and alpha2 proteins combine to form a heterodimer This heterodimer binds to di erent DNA sequences to the monomers, and represses haploid specific genes One of the repressed genes is alpha1, so alpha-specific genes are no longer transcribed Another repressed gene is RME1. RME1 represses meiosis genes, so when this is no longer transcribed, meiosis is possible Eukaryotic enhancers can have complex interactions between TFs Several proteins regulate human interferon-beta. This is important in viral defense A number of proteins bind to the interferon-beta enhancer to form an enhancesome. The proteins co-operate in binding to form a stable complex These bindings are aided by an architectural DNA-binding protein - HMG-I(Y) HMG-I(Y) induces DNA bending, which helps other factors bind. The DNA is not bent in the final complex Once the enhaceosome is complete, the interferon-beta gene is activated Nuclear receptor TFs respond directly to hormonal signals Pa erns of gene cellular expression are constantly changing in response to developmental needs and environmental cues Signal cascades are o en the result of changing conditions, with transcriptional changes one of the outcomes Nuclear receptor proteins respond specifically to e ectors (such as sex hormones). Nuclear receptors have a DHA binding domain and a ligand binding domain Binding a ligand (like a hormone) induces a conformational change in the nuclear receptor, allowing it to recruit di erent co-repressors or co-activators More complex networks can be involved. A nuclear receptor outside the nucleus may only be able to enter the nucleus when bound to the ligand Many other TFs respond to multi-step signaling cascades An example of a multi-step cascade involves NF-kB, which is important in mammalian immune responses NF-kB is a heterodimer of p50 and p65 In unstimulated cells, NF-kB is held in the cytoplasm by I-kB I-kB binds to the region of NF-kB that targets it to the nucleus Infection triggers a cascade that activates I-kB kinase, and I-kB is phosphorylated Phosphorylated I-kB ubiquitinated by E3 ubiquitin ligase, and is thus targeted for degradation by the proteasome This exposes the nuclear localization signal of NF-kB, and it can move to the nucleus and activate transcription Gene silencing can be achieved by chromatin structure changes Hox genes are a family of transcription factors that control the body plan in animals Hox genes are arranged in the genome in the same order as their expression along the body axis Cells need to keep Hox genes switched o in parts of the body where they should not be expressed. Polycomb repressive complex proteins achieve this The polycomb repressive complexes (PCR1 and PCR2) can silence stretches of Regulation of Transcription Side notes: chromatin in animal genomes PCR2 deposits and binds to H3K27me3 at nucleosomes, spreading the H3K27me3 mark across a “domain” of chromatin PCR1 then recognizes the H3K27me3 histone mark, and compacts chromatin into a repressive form The spreading of the Polycomb repressive complex across chromatin is halted by “insulator” transcription factors. CTCF plays this role in vertebrates Gene silencing can be achieved via DNA methylation DNA modifications can mediate transcriptional silencing, as in IGF2 and H19 IGF2 produces a growth factor needed by a developing embryo. H19 makes a non-coding RNA that may play a role in cancer. Both genes are on chromosome 11 IGF2 is expressed only on the maternal, and H19 on the paternal chromosome This is due to methylation at the insulator control region (ICR). The protein CTCF can only bind to ICR when ICR is unmethylated (as in the maternal chromosome) In the maternal chromosome, when CTCF is bound to ICR, proteins bound to the enhancer can stimulate transcription at H19 but are blocked from IGF2 In the paternal chromosome, CTCF cannot bind so IGF2 is not blocked and can be transcribed. H19 is also methylated, and this inhibits transcription Transcription is thought to be mediated by looping of the DNA, and determined by access of blocking of enhancer bound proteins Failure of correct imprinting can lead to Beckwith-Wiedemann syndrome children are larger than normal at birth and are prone to cancer Unlike with CTCF, methylated DNA can recruit specific proteins. Methyl-binding domains bind specifically to methyl-cytosine MeCP2 is an example of this. It binds to methylated DNA and recruits Sin3A, a transcriptional co-repressor that contains a histone deacetylase MeCP2 represses a number of human genes - mutations in this protein can cause Re syndrome This syndrome is primarily found in girls, because MeCP2 is on the X chromosome. Males have only one X chromosome, and most o en do not survive a pregnancy. Females would have one mutant and one good copy The syndrome has a number of symptoms, including some similar to autism What we don’t know (yet) about eukaryotic gene regulation What DNA pa erns TFs bind Why TFs bind some target sites and not others Which proteins/complexes each TF can interact with Whether there is a “grammar” between TFs at regulatory sites Why enhancers regulate some nearby genes and not others How enhancers regulate promoters across large distances What the role local protein concentrations might play