Gene Regulation in Eukaryotes Dr. Syahril Abdullah Medical Genetics Laboratory syahril@medic.upm.edu.my
Lecture Outline 1. The Genome 2. Overview of Gene Control 3. Cellular Differentiation in Higher Eukaryotes 4. The Regulation of Gene Expression 4.1. Genomic Level Control 4.2. Transcriptional Level Control 4.3. mrna Processing & Nuclear Transport Control 4.4. Translational Level Control 4.5. Post-Translational Level Control 5. Review A darn difficult topic You better stay awake! syahril@medic.upm.edu.my
The Genome 1. Bacteria e.g. E. coli has genome of 4 x 10 6 base pairs - 3000 gene products 2. Human genome: 3,200,000,000 (3 billion) bp (haploid) - but only 20,000-25,000 gene products - i.e. 80-90% of human genome do not have direct genetic function!! - hence redundancy of eukaryotic genome Organism Type Organism Genome Size (bp) Amoeba Amoeba dubia 670,000,000,000 Nematode Caenorhabditis elegans 100,300,000 Insect Apis mellifera (honey bee) 1,770,000,000 Fish Protopterus aethiopicus 130,000,000,000 C-value Enigma there is no correlation between complexity of an organism and its genome size!! syahril@medic.upm.edu.my
Overview of Gene Control 1. There are many different cell types in a multicellular organism (white blood cells, neurons, epithelial cells etc) 2. Each cell type arises from the selective expression of a subset of genes in the genome. 3. In many cases, the genetic program that predetermines a cell to be a certain cell type can be re-programmed to become another type of cell. 4. In cloning Dolly the sheep, the researcher took the nucleus from a lamb s udder and placed it into an egg of which the nucleus has been removed - the transplanted nucleus regenerated the whole lamb.
Overview of Gene Control 5. Many biochemical processes are common to all cell types, and thus a majority of genes are expressed in all cell types (e.g., glycolytic pathway enzymes, actin, etc.) 6. Other biochemical processes are specific to certain cells (e.g. hemoglobin in red blood cells). 7. In many cases, these tissue-specific genes are highly expressed in one or a few types of cells and not expressed at all in others.
Cellular Differentiation in Higher Eukaryotes 1. Each mammalian cell contains the same complete set of genome, regardless of which tissues or organs they are from (two copies except haploid cells). Nucleus contains all the necessary information, encoded in DNA, to control the formation of a whole organism 2. Yet different types of mammalian cells express widely different proteins even though each cell has the same complement of genes
Cellular Differentiation in Higher Eukaryotes 3. In addition, the same type of cells can have different patterns of protein synthesis during different developmental stages, for example the globin genes Different members of the globin gene family are are transcribed at different stages of human development
The Regulation of Gene Expression 1. Genomic Level Control - involves silencing or expression at chromatin structure or at DNA level. 2. Transcriptional Level Control - involves turning on or off the gene expression - most important point of control for most genes 3. mrna Processing & Nuclear Transport Control - controlling how the primary RNA transcript is spliced or processed - some RNAs are selectively transported to the cytoplasm 4. Translational Level Control - selecting which mrnas are translated by ribosomes - control of mrna stability 5. Post-Translational Processing - at level of protein - may be modified by various mechanisms like phosphorylation, ligand binding and etc. - affected by the rates of protein degradation, or its subcellular localization
1. Genomic Level Control 1. There are transcriptionally active and inactive regions through out the genome. 2. How are these regions controlled? A. Methylation of cytosine residues in DNA B. Histone modifications i. Histone Acetylation ii. Histone Methylation C. Chromatin Remodeling 3. These are the types of Epigenetics What is epigenetics? Changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence, hence the name epi- (Greek: over; above) -genetics. Changes may remain through cell divisions for the remainder of the cell's life and may also last for multiple generations.
1. Genomic Level Control : (A) Methylation of Cytosine in DNA a. CpG rich region is a short stretch of DNA in which the frequency of CG sequence is higher than other regions in the genome (p=phosphodiester bond). b. 60-90% all all CpGs are methylated in mammals c. Unmethylated CpGs are known as CpG island located in promoter regions d. DNA methylation can switch off gene expression i. By impeding the binding of transcriptional proteins (i.e. RNA pol, transcription factors). DNA methyltransferase ii. Methylated DNA bound by methyl-cpg-binding domain proteins (MBDs) recruits additional proteins.remodel histones next slides e. Active gene (expressed gene) is undermethylated; Inactive (silent) gene is hypermethylated f. Loss of methyl-cpg-binding protein 2 (MeCP2) = Rett syndrome MBD2 causes transcriptional silencing of hypermethylated genes in cancer
1. Genomic Level Control : (B) Histone Modifications i. Histone Acetylation 1. Histone acetyltransferase (HAT) acetylate histone proteins = genes transcriptionally active 2. From previous slide: MBDs bound to methylated CpG, recruits histone deacytelases (HDAC) takes away the acetyl group = genes transcriptionally inactive.
1. Genomic Level Control : (B) Histone Modifications Transcriptionally inactive Transcriptionally active Chromatin: DNA + Histones i. Euchromatin = loosely packed, active genes ii. Heterochromatin = condensed region, genes transcriptionally silent. At centromeres
1. Genomic Level Control Transcription Transcription Factors RNA Pol Acetylation Association between CpG methylation and histone acetylations DNA Methyltransferase 5-methyl-C 1. Silencing due to the chromatin compaction. 2. Interfere with the entry of transcription factors. Methyl CpG Binding Proteins Histone Deacetylase NO Transcription Deacetylation Transcription factors Chromatin Compaction Transcriptional Silencing
1. Genomic Level Control : (B) Histone Modifications ii. Histone Methylation 1. Addition of methyl groups to the tail of histone proteins 2. Activation or repression depending on which amino acids in the tail are methylated. 3. For activation of transcription: - Addition of methyl at lysine 4 in the tail of H3 histone protein (H3K4me3) - Frequently found in promoters of transcriptionally active genes. (NURF) = Nucleosome Remodeling Factor H3K4me 4. For repression of transcription - Addition of methyl at lysine 9 in the tail of H3 histone protein (H3K9me3) H3K9me
1. Genomic Level Control : (C) Chromatin Remodeling 1. Some transcription factors & regulatory proteins alter chromatin structure without altering the chemical structure of the histones directly. 2. Known as: Chromatin Remodeling Complex. 3. They bind directly to particular sites on DNA and reposition nucleosomes, allowing trascription factors to bind to promoters.
1. Genomic Level Control : DNase I Hypersensitivity How do we know if the genes are transcriptionally active? The regions around the genes become highly sensitive to the action of DNase I Regions known as: DNase I Hypersensitive Sites Develops about 1kb upstream from the transcription start site Indicates that these regions adopt a more open configuration.
1. Genomic Level Control Epigenetic Inheritance? How histone modifications, nucleosome positioning & other types of epigenetic marks might be maintained is still unclear
2. Transcriptional Level Control Promoter Start of translation: AUG Enhancers/ Silencers Upstream Elements TATA Box -1 kb -25/-30 bp +1 bp Promoters: A DNA sequence to which RNA Pol binds prior to initiation of transcription. Contains a sequence called TATA box (7 bp consensus sequence 5 -TATA[A/T]A [A/T]-3 ). Enhancers: To stimulate/increase the activity of the promoters Orientation and position independent Silencers: Inhibits transcription Also orientation and position independent Transcription Factors (TFs): Bind to regulatory DNA sequences (promoters, enhancers) to regulate transcription Two types: (i) Basal TFs (eg. TFIIA, TFIIB)- bind at promoters, assisting RNA pol (ii) Specific TFs (eg. Sp1, C-Jun) bind at specific enhancers
2. Transcriptional Level Control
2. Transcriptional Level Control Hormonal Effects on Enhancer Human metallothionein protein 1. Regulation of zinc (Zn) & copper (Cu) in blood, detoxification of heavy metals, function of immune system, neuronal development. Synthesized in kidney and liver. 2. Usually expressed at very low level 3. Gene expression can be activated by cadmium(cd), copper(cu) ions or by glucocorticoid hormone. When glucocorticoid hormone is released, it binds to the glucocorticoid receptor (a kind of specific TF) protein Glucocorticoid receptor protein (+glucocorticoid) recognizes a specific enhancer called Glucocorticoid Response Element (GRE) in the metallothionein gene and binds to it -- this activates expression of the metallothionein gene. Response elements function in response to transient increase in the level of a substance or a regulatory hormone
2. Transcriptional Level Control Insulator 1. Also known as boundary element 2. What it is? DNA sequences that block or insulate the effect of enhancers in positiondependent manner
3. mrna Processing and Nuclear Transport Control 1. Splicing: The process of cutting the pre-mrna to remove the introns and joining together the exons. 2. Alternative splicing: is a process that occurs in which the splicing process of a pre-mrna transcribed from one gene can lead to different mature mrna molecules and therefore to different protein." Fibronectin Gene Exon EIIIB Exon EIIIA Primary mrna transcript of fibronectin gene 5 3 Fibroblast mrna Liver mrna - exons EIIIA and EIIIB are spliced out in liver mrna transcript A single gene can code for two or more related proteins, depending on how the exons/ introns are spliced
3. mrna Processing and Nuclear Transport Control 1. Speed of Transport of mrna Through the Nuclear Pores Evidence suggests that this time may vary. 2. Longevity of mrna mrna can last a long time. For example, mammalian red blood cells eject their nucleus but continue to synthesize hemoglobin for several months. This indicates that mrna is available to produce the protein even though the DNA is gone. Ribonucleases are enzymes that destroy mrna. mrna has noncoding nucleotides at either end of the molecule contain info about the number of times mrna is transcribed before being destroyed by ribonucleases. Poly A tail stabilizes mrna transcripts. Hormones can stabilize certain mrna transcripts Prolactin Prevents Digestion Gene for Casein mrna Casein Milk DNA Ribonuclease Ribonuclease Digest
4. Translational Level Control 5 Untranslated Region (5 UTR) Starts from transcription start site to just before the initiation codon (ATG) Contains sequence that regulate translation efficiency i. Binding site for proteins that may effect the translation e.g. Iron responsive elements (also in 3 UTR) regulate gene expression in response to iron. ii. Kozak sequence RccAUGG, where R is a purine (A or G) 3 bases upstream of the start codon, follow by another G. Translation more efficient with Kozak sequence. 3 Untranslated Region (3 UTR) Starts from stop codon, end before poly A tail. Contains regulatory sequence for efficient translation i. For cystoplasmic localization of mrna ii. Binding site for : SECIS elements direct ribosome to translate codon UGA as selenocysteines. MicroRNA (a type of RNAi)
4. Translational Level Control A bit about RNA interference (RNAi) 1. From DNA, transcribed but not translated 2. About 30% of human genes regulated by RNA interference 3. In eukaryotes, fungi, plants, animals RNAi syahril@medic.upm.edu.my
4. Translational Level Control : RNAi Mechanisms 1. RNA Cleavage 2. Inhibition of Translation 3. Transcriptional Silencing RISC: RNA-induced silencing complex RITS: RNA-induced transcriptional silencing
5. Post-Translational Processing These mechanisms act after the protein has been produced 1. Protein cleavage and/or splicing. The initial polypeptide can be cut into different functional pieces, with different patterns of cleavage occurring in different tissues. In some cases, different pieces may be spliced together. e.g. Bovine proinsulin is a precursor to the hormone insulin. It must be cleaved into 2 polypeptide chains and about 30 amino acids must be removed to form insulin.
5. Post-Translational Processing 2. Chemical modification. Protein function can be modified by addition of methyl, acetyl, alkyl, phosphoryl, or glycosyl groups. E.g. How can phosphorylation control enzyme activity? Addition of phosphate causes conformational changes to the protein. Opens up the active site for catalytic process.
Review
The End syahril@medic.upm.edu.my