Specificity of gene expression: “different folks, differentA cell typically strokes” expressesonly a fraction of its genes, and the different types of cellsin multicellular organisms arise because different sets of genes are expressed.
Eukaryotic Gene RegulationLevels of eukaryotic generegulation: Genome (amplification or rearrangement of DNA segments, chromatin remodeling: decondensation/ condensation and DNA methylation). Transcription. Processing (and nuclear export) of RNA. Translation (and targeting) of protein. Posttranslational events (folding and assembly, cleavage, chemical group modifications and organelle import/secretion). Degradation of mRNA and proteins http://highered.mcgraw- hill.com/olc/dl/120080/bio31.swf
GENOMIC CONTROL Yeast mating-type switching depends upon the swapping of genetic cassettes to alter the DNA sequence. Saccharomyces cerevisiae has mating- types (sexes): α or a. Chromosome III contains three separate copies of the mating-type information. The HML α and HMR a loci contain complete copies of the α and a The cells actual mating type is determined forms but the by the allele present at the MAT locus. transcription of these When a cell switches mating types, the α or loci is inhibited by the a DNA at the MAT locus is removed and products of gene the SIR replaced by a DNA "cassette" copy of the gene. alternative mating type DNA.
Active genes are specifically attached to thenuclear matrix by AT-rich DNAsequences calledmatrix-associated regions (MARs), which arerecognized by an enzyme- topoisomerase that unwind the DNA helix
Different chromatin remodelingcomplexes disrupt and reformnucleosomes. The same complex might catalyze both reactions. The DNA-bindingproteins could be involved in gene expression, DNA replication, or DNA repair.
Normal chromatin blocks the association of transcription factors and polymerase II withthe DNA. Chromatincan enter a "silent" state or can be converted to a "poised" state through histone acetylation.
Transcriptional activation by histone acetylation. p300/CBP can interact with a variety of transcriptional regulators such as pCAF (a histone acetyltransferase) and TBP (which recognizes the promoter). During transcription, they are all assembled at the promoter region. Histone acetylation by p300/CBP, pCAF and TAFII250 facilitates the transcription. Transcriptional repression by histone deacetylation. The repressor Mad/Max dimer interacts with SIN3 (or N- CoR, SMRT, etc.) whichrecruits histone deacetylase (HDs) to repress transcription.
CpG islands (CG clusters) occur in 40,000 regions near 5’ ends of genes. They are typically unmethylated; are thought to indicate the positions of ‘housekeeping’ genes. DNA methylase methylate CG regions on DNA. creating a “5th base" in DNA, 5-methylcytosine. This base is made enzymatically after DNA is replicated, at which time about 5% of the cytosines in mammalian DNA are converted to 5-methyl cytosine.
Both methylation and acetylation provide means for maintaining the state of activity of developmental genes even after the original signals for activation or repression have disappeared Examples: globin promoters in RBC are unmethylated, globin promoters are highly methylated in other cells.
How is methylation involved in repressing genes? Methylated DNA is preferentially bound by histone H1, the histone that associates nucleosomes into higher-order folded complexes. Histone deacetylases aggregate in methylated regions of DNA, causing them to bind tightly to DNA, and form a stable nucleosome. Acetylation of histones reduces their affinity to DNA and cause the nucleosomes to disperse. This opens up chromatin structure for binding with the transcription complex.
How DNA methylation patterns are faithfully inherited. During replication, the DNA template strand retains the methylation pattern, while the newly synthesized strand does not. However, the enzyme DNA cytosine-5- methyltransferase has a strong preference for amethylated strand, and when it sees a methyl-CpG on oneside of the DNA, it methylates the new C on the other side.
Differential methylation in germ cells is implicated in genomic imprinting, the marking of a gene as either maternal or paternal. Paternal 15q- causes Prader-Willi syndrome (mild mental retardation, obesity, small gonads, short stature) Maternal form causes Angelman syndrome (severe mental retardation, seizures, lack of speech, inappropriate laughter).
In human females, random X-inactivation is due to unmethylation of the Xist gene, which produces a XIST transcript that coats the X- chromosome. This produces a XIST-Barr body complex that inactivates the chromosome.
Since DNA methylation is not encoded in the DNA sequence itself, it is called an EPIGENETIC modification. DNA methylation is therefore a form of cellular memory.
Epigenetic marks are ERASED during gametogenesis and reset to ensure appropriate gene activity. Old marks are purged and new tags are established in a process called PROGRAMMING. However, some methylated epigenomes can escape purging during gametogenesis. These genes are inherited by mature gametes and are carried into the next generation as GENOMIC IMPRINTS. In a pregnant mother, 3 generations are directly exposed to the same environmental conditions at the same time. Germ cells thus carry the effects of gorgeous lola’s lifestyle!
Bisphenol A in baby’s bottle is linked to obesity. Causes demethylation that reprograms the hypothalamus resulting to increased appetite Supplementing the mothers diets with methyl-donating substances (folic acid, Mice are GENETICALLY vitamin B12) and identical but genistein in soy products EPIGENETICALLY counteract the reduction different. in DNA methylation caused by bisphenol A. The yellow mouse is exposed to bisphenol A
TRANSCRIPTIONAL CONTROL Different subsets of genes are transcribed in different tissues. Tissue specific gene regulation is responsible for these differences. DNA microarrays are used to profile gene expression patterns in various cells.
Differences between the populations of mRNA sequences in thecytoplasm of different kinds of cells reflect corresponding differences in nuclear RNA populations which result from differential transcription of genes. cDNA probes for tissue-specific transcripts are prepared byeliminating from the tissues mRNA population the molecules also found in another tissue and reverse-transcribing the remaining mRNA.
The core promoter is where the general transcription factors and RNA polymerase assemble for the initiation of transcription. Presence of basal transcription factors is not sufficient for the activation of the promoter. DNA-binding proteins regulate genes either by repression or activation. Regulatory sequences (enhancers or silencers) serve as binding sites for these proteins, whose presence on the DNA affects the rate of transcription initiation.
A Model for Enhancer Action An enhancer is brought close to the core promoter by a looping of the DNA. The influence of an enhancer on the promoter is mediated by regulatory transcription factors called activators. The activator proteins bind to the enhancer elements, forming an enhanceosome. Bending of the DNA brings the enhanceosome closer to the core promoter. TFIID is in the promoters vicinity. The DNA-bound activators interact with specific coactivators that are part of TFIID. This interaction facilitates the correct positioning of TFIID on the promoter. The other general transcription factors and RNA polymerase join the complex, and transcription is initiated.
Enhancers are modular. There are various DNAelements that regulate temporal and spatial geneexpression, and these can be mixed and matched. Whether the gene is transcribed or not will depend on the combination of transcription factors present.
The gene for the protein albumin is associated with an array of regulatory DNA elements. Cells of all tissues contain RNA polymerase and the general TFs, but the setof RTFs available varies with the cell type. Liver cells contain a set of RTFs that includes the factors for recognizing all the albumin gene control elements. Whenthese factors bind to the DNA, they facilitate transcription of the albumin gene at a high level. Brain cells have a different set of RTFs, which does not include all the ones for the albumin gene. In brain cells, the transcription complex can assemble at the promoter, but not very efficiently. The result is that brain cells transcribe the albumingene only at a low level.
The DNA response sequences that bind transcription factors are often comprised of inverted repeat elements. Reading the sequence of the glucocorticoid response element in the 5’- 3’ direction from either end yields the same DNA sequence (5’-AGAACA -3’). The thyroid hormone element contains the same inverted repeat sequences as the estrogen element but the 3 bases that separate the 2 copies of the sequence in the estrogen element are absent.
The glucocorticoid receptors activate Binding the steroid gene transcription. Cortisol, a causes the release of an hydrophobic steroid hormone, can inhibitory protein and diffuse through a plasma membrane activates the then bind to the intracellular glucocorticoid receptor glucocorticoid receptor. molecules DNA binding site. The glucocorticoid receptor molecule then enters the nucleus and binds to a glucocorticoid response element in DNA which causes a 2nd glucocorticoid receptor molecule to bind to the same response element. The resulting glucocorticoid receptor dimer activates transcription of the target gene.
Transcription factors are proteins that bind to the enhancer or promoter regions and interact such that transcription occurs from only a small group of promoters in any cell. Most have: a.DNA- binding domains that bind to promoter or enhancer regions of DNA of specific genes b.trans-activating domain that binds RNA polymerase II or other transcription factors to regulate amount of mRNA that the gene produces c.protein-protein interaction domain that allows the transcription factors activity to be modulated by TAFs or other transcription factors.
Generegulatory proteins contain structuralmotifs that can read DNAsequences
Different base pairs in DNA can be recognized fromtheir edges without the need to open the double helix.Regulatory proteins make contact with major groove .
Determining DNAsequence recognizedby a regulatory protein
A DNA fragment is labeled at one end with 32P. The DNA is cleaved that makes random, single stranded cuts.After the DNA denaturation, the resultant fragments from thelabeled strand are separated on a gel and detected byautoradiography. The pattern of bands from DNA cut in the presence of a DNA-binding protein is compared with that from DNA cut in its absence. When protein is present, it covers the nucleotide at its binding site and protects their phosphodiester bonds from cleavage. As a result, those labeled fragments that would otherwise terminate in thebinding site are missing, leavinga gap in the gel pattern called a "footprint.“
Several structural motifs are commonly found in the DNA-binding domainsof regulatory transcription factors. The parts of these domains that directly interact with specific DNA sequences are usually α helices (or recognition helices) which fit into DNAs major groove. The helix-turn-helix motifcontains 2 α helices joined by a short flexible turn. The zinc finger motif has an α helix and a 2-segment, antiparallel ß sheet, all held together by theinteraction of 4 cysteine residues (or 2 cysteine & 2 histidine residues) witha zinc atom. Zinc finger proteins normally contain a number of zinc fingers.
The leucine zipper motif contains an α helix that has a regulararrangement of leucine residues that interacts with a similar region in a 2nd polypeptide to coil around each other. The helix-loop-helix motif contains a short and long helix connected by a polypeptide loop that interacts with a similar region on another polypeptide to create a dimer, e.g. human TF MAX, lac and trp repressor.
The homeodomain is a helix-turn-helix DNA- binding domaincontaining 3 α helices encoded by a 180 bp homeobox. This was originally found inhomeotic genes which control the establishment ofpattern in an organism such as the spinalcolumn in vertebrates.
Superclass: Basic Domains Families AP-1(-like) components: Jun, Fos, GCN4, CRE-BP/ATF Leucine zipper factors CREB: CREB, ATF-1, (bZIP) C/EBP-like factors and 4 other families Helix-loop-helix factors Myogenic transcription factors (bHLH) and 8 other families Helix-loop-helix / leucine Cell-cycle controlling factors:zipper factors (bHLH-ZIP) Myc, Max, and 1 other family NF-1 RF-X bHSH
Superclass: Zinc-coordinating DNA-binding domains Steroid hormone receptors Cys4 zinc finger of Thyroid hormone receptor-like factors: nuclear receptor type RAR, RXR, Vitamin D receptor, PPAR, Knirps 4 families: WT-1 in the kidney, Krox20 in Cys4 zinc fingers the rhombomeres of the hindbrain Ubiquitous factors: TFIIIA, Sp1 Developmental / cell cycle regulators: Cys2His2 zinc finger Krüppel, Hunchback domain 3 other families involved in cytoskeletal organization, organ development and oncogenesis Cys6 cysteine-zinc Metabolic regulators in fungi cluster Zinc fingers of 2 familiesalternating composition
Superclass: Helix-turn-helix Homeodomain only: Antp, Ubx, Engrailed, Eve POU domain factors: Oct-1, Oct-2 Homeodomain (activate IgG genes) 2 other families implicated in craniocaudal segmentation of the body. Pax1-9: Pax6 in the eye, Pax3 in the Paired box developing somiteFork head / winged Developmental regulators: Forkhead helix 2 other families: HNF3Heat shock factors MybTryptophan clusters 2 other families TEA domain
Superclass: β-Scaffold Factors with Minor Groove Contacts Rel/ankyrin: NF-kappaB1RHR (Rel homology region) 2 other families STAT p53 Regulators of differentiation: MADS box Agamous, AP1, Deficiens, AP3 2 other familiesbeta-Barrel alpha-helix TFs TATA-binding proteins TBP TDF-SRY, TCF factors HMG activated by the Wnt pathwayHeteromeric CCAAT factors GrainyheadCold-shock domain factors Runt
Superclass: Other Transcription Factors Copper fist proteins HMGI(Y) Rb - retinoblastoma Pocket domain 1 other family E1A-like factors E1A AP2AP2/EREBP-related factors 2 other families The most recent update of the transcription factor database lists 2785 entries. Many of these are homologous proteins from different species, nevertheless this number is indicative of the vast number of transcription factors now known thatregulate the expression of eukaryotic genes. Majority function as ligands in cell signaling pathways.
Alternative splicing makes it possible for the same gene to produce several different mRNAs, by splicing together different combinations of exons from the primary transcript. Different proteins formed from the same gene are called splicing isoforms.
Genes coding for the human antibody heavy chains are created by DNA rearrangements involving multiple types of V, D and J segments. Initially, the DNA of the immune cells is arranged as tandem arrays of V, D and J regions DNA excision randomly removes several D and J segments to place individual D and J sequences side by side. A second random excision removes several V and D segments to join a V section to the others to form a VDJ segment. After transcription, the sequences separating the VDJ segment from the C segment are removed by RNA splicing.
The antibody protein immunoglobulin M (IgM) exists in 2 forms, as secreted IgM andmembrane-bound IgM. These molecules, encoded by a single gene, differ in their heavy chain’s carboxyl ends. The IgM gene has 2 possible poly(A) addition (termination) sites and a number of exons that can produce 2 alternative forms. The plasma membrane- bound form contains a transmembrane anchor which is encoded by exons 5 and 6. If a splice junction within exon 4 is used, exons 5 and 6 (carrying the anchor) are added togenerate the IgM heavy chain. The secreted product is produced when the exon 4 splice is not made and these transcripts are terminated just after exon 4.
Developing RBC synthesize hemoglobin (4 globin polypeptides and a heme prosthetic group). The heme-controlled inhibitor (HCI)protein regulates hemoglobin synthesis inresponse to the presence of heme. Whenpresent, heme binds to inactivate the HCI. When heme is absent, the HCI is active. Active HCI functions as a kinase that catalyzes the phosphorylation of eIF2, a key TF. P-eIF2 is inactive; it cannot When heme iscombine with methionyl tRNA and GTP to presentform the translation initiation complex. In translation of the absence of heme, translation of all the mRNAmRNA in the cell is inhibited. The effect is proceeds. Newlyon globin synthesis because globin mRNA made globinsconstitutes most of the developing RBC’s combine with mRNA. heme to form hemoglobin molecules.
Translation of ferritin is activated in the presence of iron.Translation is inhibited by binding of the IRE-binding proteinto the hairpin structure of an iron response element (IRE) in the 5’UTR leader sequence of ferritin mRNA. When ironbinds to IRE-binding protein, it contorts into a conformation that does not recognize the IRE. When iron is available, ribosomes can assemble on the mRNA and proceed to translate ferritin. The hairpin does not interfere with the ribosome activities.
Degradation of the transferrin receptor mRNA (required for iron uptake) is also regulated by the allosteric IRE-bindingprotein. Transferrin receptor mRNA has an IRE in its 3’ UTR. When intracellular [iron] is low, the IRE-binding proteinremains bound to the IRE which 1) protects the mRNA from degradation and 2) allowing more transferrin receptor protein to be synthesized. When intracellular [iron] is high, iron binds to the IRE-binding protein, it releases the IRE and the mRNA can be degraded.
By RNA interference, short RNAs can lead to silencing the expression of genes that contain complementary sequences in their mRNA. A complex of dsRNA is cleaved into short fragments of 21-22 bp in length by theribonuclease Dicer. The fragments are siRNAs (short interfering RNAs). ThesiRNAs bind to the RISC (RNA-inducedsilencing complex). One of the strands of siRNA is degraded. The remaining single-stranded siRNA , complexed with the RISC can then bind to complementary mRNA. If a perfect or near perfect match, the mRNA is cleaved. In addition, the RISC-siRNA complex can enter the nucleus, binds the genomic sequence and initiates a DNA methylation based chromatincondensation inactivation of the gene.
microRNAs (miRNAs) are gene products that are 21-22 nucleotides in length. The 10miRNAs are transcribed, form hair- pin structures and are cleaved by Drosha to make precursor microRNAs (70 nucleotides in length). The pre-miRNAs are exported to the cytoplam where they are cleaved by Dicer into the 21-22 nucleotide mature microRNAs. The miRNAs formRNP complexes with mRNAs. If the match is exact, the mRNA is destroyed, similar to siRNA mechanisms. If the match is less-than-exact, then binding (usually of several miRNAs) inhibit translation. Genes for miRNAs make up 0.5-1.0% of the total number of genes in multicellular organisms, i.e. 200-250 miRNA genes in humans.
Several changes can still take place that determine whether or not the protein will be active. In the ER, the protein becomes further processed (e.g. formation of disulfide bonds, glycosylation, phosphorylation). The folded proteins are carried in vesicles to the Golgi apparatus where further processing of the carbohydrates occurs, and then to exocytotic vesicles where they are released.
Proteins can be marked for destruction by the addition of ubiquitin. 1) A protein targeted for degradation is bound at its N-terminus by a ubiquitinating enzyme complex. 2) In an ATP-dependent series of reactions, ubiquitinmolecules are sequentially attached to the proteins lysine residues. Theubiquitinating enzyme complex then detaches. 3) A proteasome degrades the ubiquitinated protein into short peptides. The ubiquitin is released and can be recycled.
Some newly synthesized proteins are inactive without the cleaving away of certain inhibitory sections. (e.g., insulin is processed from its larger protein precursor). Some proteins must be "addressed" to their specific intracellular destinations in order to function. Proteins are often sequestered in certain regions, such as membranes, lysosomes, nuclei, or mitochondria. Some proteins need to assemble with other proteins to form a functional unit. Some proteins are not active unless they bind an ion such as calcium, or are modified by the covalent addition of a phosphate or acetate group.