IBS 8102 Cell, Molecular, and Developmental Biology Differential Gene Expression January 22, 2008
Differential Gene Expression Chromatin structure Gene anatomy Gene sequences Control of gene transcription enhancers transcription factors Epigenetic processes (modification of structure of DNA rather than sequence) methylation imprinting epigenetics RNA processing
Chromatin Structure ~140 bp chromatin ~60 bp Histone lysines are protonated = net positive charge DNA PO 4 deprotonated = net negative charge electrostatic attraction ensures tight binding DNA inaccessible to polymerases, etc
Nucleosome Tails Core DNA bound to an octameric complex composed of two each of histones H2A, H2B, H3, and H4. From Luger et al, 1997. Nature 389:251 260. Histone H1 associates nucleosomes into higher order folded complex
Chromatin Acetylation Transcriptional Regulation: 1. Packing prevents access CH 3 2. Acetylation ( ) state of histones controls DNA binding C O acetylated histone (histone acetyltransferase; HAT) destabilizes = transcription allowed e.g. acetylation of K14 and K9 lysines on tail of histone H3 correlated with transcriptional competence deacetylated histones (histone deacetylases; HDAC) stabilizes = transcription repressed 3. Histone methylation (CH 3 ) transcriptional repression methylation of K9 lysine on H3 associated with heterochromatin Multiple histone regulatory modifications may occur at once; work together to change the behavior of the chromatin dynamic systematic = Histone Code reproducible
Chromatin Configuration John H. Frenster Heterochromatin remains tightly condensed throughout most of the cell cycle replicates later Euchromatin active chromatin
Anatomy of the Gene core promoter transcription initiation site region transcribed region 5 TATAT 3 +1 upstream downstream Transcription produces heterogenous nuclear RNA (hnrna)
hnrna Processing promoter exon 1 intron 1 exon 2 intron 2 termination region 7 methyl Gppp cap primary transcript gene transcription, 5 capping 3 cleavage, polyadenylation cleavage factor cleavage signal AAUAA poly(a)polymerase 7 methyl Gppp AAAAAAA(n) splicing 7 methyl Gppp exon 1 exon 2 AAAAAAA(n) = mature mrna, ready to be transported to the cytoplasm and translation
Human β globin Sequence
Production of β globin
Promoters and Enhancers RNA polymerase II binds to the promoter region at the TATA box however, Pol II cannot initiate transcription alone various proteins bind to regulatory sequences upstream and downstream of transcription initiation site promoter region binds Basal Transcription Factors promoter region 5 3 basal transcription factors facilitate Pol II binding and activity
Eukaryotic Transcription Initiation Complex (AKA Preinitiation Complex) Basal (General) Transcription Factors: small nuclear proteins bind to promoter region constitutive, ubiquitous bind sequentially binding of individual TFs mediated by small proteins TBP associated factors (TAFs) mediator complex (~25 proteins)
Eukaryotic Transcription Initiation Complex 1. TFIID complex binds to the TATA box through its TATA Binding Protein (TBP) subunit 2. TFIID is stabilized by TFIIA 3. TFIIB and TFIIH join the complex on the TATA box; TFIIE and TFIIF associate with RNA polymerase II
Eukaryotic Transcription Initiation Complex 4. RNA polymerase II is positioned by TFIIB, and its carboxy terminal domain (CTD) is bound by TFIID 5. The CTD is phosphorylated by TFIIH and is released by TFIID; RNA polymerase II can now transcribe mrna
Stabilization of Transcriptional Initiation Complex by TAFs TBP TATA binding protein TAF(s) TBP associated factors SP1, NTF 1 = Transcription factors
TAF Histone Acetyltransferase Activity TBP TATA binding protein TAF(s) TBP associated factors
Enhancers Enhancers are cis acting regulatory elements cis (same or same side); elements that reside on the same DNA strand; e.g. DNA sequences trans (other side); elements that originate from another DNA strand, e.g. regulatory proteins Enhancers: DNA sequences that regulate gene expression by affecting the transcription initiation complex on the promoter. Function bind specific regulatory proteins (i.e. specific Transcription Factors) TFs generally interact with mediator complex proteins recruit RNA polii Location highly variable with respect to the transcribed portion of the gene. upstream (5 ), downstream (3 ), or within transcribed region close proximity or as many as 10 6 bp away
Enhancers Enhancers differ from promoters: 1) need a promoter to work 2) can work at a distance 3) can work in reverse orientation
Enhancer Generalizations 1. Most gene transcription requires enhancers. 2. Enhancers are the major determinants of differential transcription in cell types and through developmental stages. 3. There can be multiple signals (e.g. multiple enhancer sites) for a given gene, and each enhancer can be bound by more than one transcription factor (not at the same time). 4. Transcription is regulated by the interaction of transcription factors bound to enhancers and the transcription initiation complex assembled at the promoter. 5. Enhancers are combinatorial. Various DNA sequences regulate temporal and spatial gene expression; these can be mixed and matched. 6. Enhancers are modular. A gene can have several enhancer elements, each of which may turn it on in different sets of cells. 7. Enhancers generally activate transcription by remodeling chromatin to expose the promoter, or by facilitating the binding of RNA polymerase to the promoter by stabilizing TAFs. 8. Enhancers can also inhibit transcription (aka Silencers).
Transcription Factors Proteins that bind to enhancer or promoter regions activate or repress transcription Most bind to specific DNA sequences (e.g. enhancers) Transcription factors are grouped together in families, based on structural similarities families share common framework in DNA binding sites slight differences in binding sites cause differences in recognition Arabidopsis currently 41 TF families listed eukaryotes > 75
TF/DNA Interactions estrogen receptor zinc finger helix loop helix leucine zipper
TF/DNA Interactions homeodomain
T box Transcription Factor
NFkB/Rel Transcription Factor
Transcription Factor Domains Three major domains: 1. DNA binding recognizes particular DNA sequence transcription factor engrailed
Transcription Factor Domains Three major domains: 1. DNA binding recognizes particular DNA sequence 2. Trans activation activates or represses transcription often involved with transcription coregulator proteins involved in binding RNA polymerase II; e.g. TFIIB, TFIIE often involved with enzymes that modify histones
Transcription Factor Domains Three major domains: 1. DNA binding recognizes particular DNA sequence 2. Trans activation activates or represses transcription often involved with transcription coregulator proteins involved in binding RNA polymerase II; e.g. TFIIB, TFIIE often involved with enzymes that modify histones 3. protein protein interaction domain promotes dimerization allows it to be modulated by TAFs or other transcription factors TBP TATA binding protein TAF(s) TBP associated factors NOTE alternate categories combine 2 and 3; also add signal sensing domain for some TFs
Transcription Factor Domains Transcription factor MITF (microphthalmia) active in ear and pigment forming cells of eye and skin; osteoclasts pigment cell specific tyrosinases expressed MITF mutation = deafness, multicolored irises, white forelock Structural Family basic helix loop helix leucine zipper Functional protein is a homodimer dimerization common among TFs The transactivating domain is located near the amino terminal when bound to a promoter or enhancer, the protein is able to bind TAF p300/cbp TAF p300/cbp is a histone acetyltransferase
Functional Classification of TFs I. Constitutively active e.g. SP1, NF1 CCAAT II. Conditionally active require activation II.A. Developmental (cell specific) expression tightly controlled, but once expressed requires no additional activation; e.g. GATA, HNF, PIT 1, MyoD, MNyf5, Hox, Winged Helix II.B. Signal dependent requires external signal for activation II.B1. Extracellular ligand dependent nuclear receptors II.B2. Intracellular ligand dependent activated by small intracellular molecules; e.g. SREBP, p53, orphan nuclear receptors II.B3. Cell membrane receptor dependent second messenger signaling cascades resulting in the phosphorylation of the transcription factor II.B.3.a. Resident nuclear factors reside in the nucleus regardless of activation state; e.g. CRDEB, AP 1, Mef2 II.B.3.b. Latent cytoplasmic factors inactive form reside in the cytoplasm, but when activated are translocated into the nucleus; e.g. STAT, R0SMAD, NF kb, Notch, TUBBY, NFAT A. H. Brivanlou et al., Science 295, 813 818 (2002)
Functional Classification of TFs A. H. Brivanlou et al., Science 295, 813 818 (2002)
Enhancer Modules Enhancers are modular: e.g. Pax6 gene expressed in the eye, pancreas, nervous system. also expressed in different genes within these tissues Within these modules, transcription factors work in a combinatorial fashion. Transcription factors operate in cascades: one stimulates the production of several others.
Combinatorial Enhancers combinatorial transcription factors NOTE enhancers can act as repressors/silencers Kaltoff, 2001
Transcription Factor Competition Kaltoff, 2001
Epigenetic Mechanisms Development is an epigenetic, not a genetic, process. Epigenetics epi above, etc. control of gene expression not involving sequence Mechanisms histone methylation DNA methylation imprinting sirna alternative RNA splicing translational control
Histone Modification Acetylation/deacetylation acetylation destabilizes nucleosome targets: lysines on histone tails Methylation stabilized nucleosome (transcription repressed) hypermethylation of H3 lysine 9 induces ectopic heterochromatin methylated histones recruit DNA methylation enzymes further stabilizes nucleosome demethylated by specific demethylating enzymes Ubiquitination vertebrates, yeast; core histones H2A, H2B, H3 destabilizes nucleosome Phosphorylation histone serine phosphorylation correlated with gene activation Others?
DNA Methylation DNA methylation stabilizes nucleosomes; prevents transcription found in all vertebrates; not Drosophila, nematodes, inverts degree of methylation is proportional to degree of transcription in adults tissues cytosine methylation occurs only in CpG sequences 1 5% of CpG in adult somatic cells are methylated in embryonic tissues and stem cells non CpGs nucleotides (CpA, CpT) are also methylated tumor suppressor genes silenced by methylation in many cancers
Embryonic DNA Methylation Methylation patterns differ in stage and tissue specific manner methylation correlates inversely with expression methylation pattern changes during development Methylation patterns are established and maintained throughout cell division by DNA (cytosine 5) methyltransferases humans: DNMT3a, DNMT3b act as de novo methyltransferases DNMT1 maintains pattern during DNA replication
Genomic Imprinting Special case of DNA methylation: alleles from maternal and paternal genome are differentially methylated methylation patterns can be distinguished based on resulting phenotypes No mammalian parthenogenesis because of imprinting; in mice: gynogenetic (two female pronuclei) embryos showed normal embryonic development, but poor placental development androgenetic (two male pronuclei) embryos showed poor embryonic development, but normal placental development Approximately 80 imprinted genes in humans; many involved with early developmental processes Imprinting seen in mammals, insects, flowering plants in insects, imprinting silences paternal genome produces males (functionally haploid)
Genomic Imprinting Imprinted alleles from maternal and paternal genomes are differentially methylated. both methylation patterns are necessary for proper development Angelman and Prader Willi syndromes: Chromosome 15 differentially methylated in males and females Deletions in either parental chromosome 15 result in predominance of reciprocal pattern Female deletion = Angelman syndrome intellectual and developmental delay speech impediment replicated by mutation of Ube3a gene (ubiquitin pathway) Male deletion = Prader Willi syndrome extreme or insatiable appetite small stature learning disabilities imprinted genes: necdin & SNRPN small nuclear ribonucleoprotein polypeptide N
Dosage Compensation Mammals, Drosophila, nematodes XX = female, XY = male; diploid XXs have double the X gene products as diploid XYs XX cell Drosophila transcription rate of male X is doubled C. elegans both Xs partially repressed ( = hermaphrodite) Mammals Inactivation of a single X chromosome in mammalian XX cells early embryo both active later embryo only one active Barr bodies
X Chromosome Inactivation Lyon hypothesis: 1. Both X chromosomes active in very early female development. 2. One X is inactivated in each cell 3. Inactivation is random 4. The process is irreversible. All progeny cells will retain the same inactivation pattern calico cat: heterozygous for coat color genes contained on early late X chromosome X chromosome inactivation
Differential RNA Processing Nuclear RNA selection more genes transcribed than processed into mrna unprocessed genes degraded in nucleus Alternate splicing ~75% of all human genes may be alternatively spliced (splice isoforms; splice variants) = one gene one protein family nrna splicing mediated through spliceosomes Spliceosomes constructed for each new hnrna small nuclear RNAs (snrna) splicing factor proteins Introns contain recognition sequences GU at 5 end splice site, AG at 3 end splice site (typical) also variable length polypyrimidine polypyrimidine tract (PPT) recruit spliceosome factors to site Cell specific splicing factors differ in ability to recognize intron sequences exon in one cells type might be recognized as an intron in another = alternative splicing
α Tropomyosin Alternative Splicing striated muscle striated muscle myoblast smooth muscle neuroblast/muscle hepatoma brain
Translational Control Differential mrna longevity stability dependent on length of poly(a) tail poly(a) length dependent on 3 UTR Selective inhibition of message translation (e.g. stored maternal messages) messages lacking 5 cap or 3 poly(a) tail are not translated (may be degraded) mrnas circularize mediated by proteins bound to 5 cap and poly(a) tail no 5 cap = no protein binding proximity of 5 and 3 ends important for ribosomal recognition, initiation stored mrnas often lack methylated cap at fertilization, methyltransferase completes cap mrna 5 methylated guanosine cap poly(a) binding protein eukaryotic initiation factors Rhoads, R. E. et al. Mechanism and regulation of translation in C. elegans (January 28, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.63.1, http://www.wormbook.org. ribosomal 40 S subunit
MicroRNAs mirnas complementary to part of one or many mrnas animals 3 UTR; plants coding region) inhibits translation or promotes degradation stem loop 70 nt NOTE Drosha/Dicer/RISC mechanism also used to process sirna