Embryology PDF Lecture Notes

Problems with the Hox Code Model - Specificity Lecture 18 Problems Hox protein target specificity If they all recognize a similar DNA sequence motif (TAAT) how can they each specify a different vertebral identity (ie; activate different genes at different times in each place)? Phenotypes not...

Problems with the Hox Code Model - Specificity Lecture 18 Problems Hox protein target specificity If they all recognize a similar DNA sequence motif (TAAT) how can they each specify a different vertebral identity (ie; activate different genes at different times in each place)? Phenotypes not always as expected If combinatorial Hox code specifies discrete addresses (positional identity), then mutation should yield a phenotype immediately withing the normal domain of Hox expression. Target Specificity Third helix of homeodomain binds to and affects major groove of target gene regulatory sequence (TAAT) - Does rest of Hox protein affect shape in subtle way to affect specificity of action? Does flanking sequence of target gene motif play a role in modulating access? Are there partner proteins that play a role in guiding specificity?  Hox Protein Structure/Partners Vary Hox proteins dimerize with: Each other ~ distance Exd/Pbx varies MEIS Hexapeptide (AKA pentapeptide region) binds partner protein Linker endows this binding site with its partner specificity Distance between homeodomain and Exd/Pbx/MEIS binding region varies Testing Different Structural Attributes Some tests: 1. Reporter gene promoter assays (tests 1000s of kb of target gene regulatory region) 2. DNase footprinting assays (tests 100s of bp of target gene regulatory region) 3. Gene Mobility Shift Assays (tests 40-60 bp of target gene regulatory region) 4. Gene disruption animal model (“knockout” mice) 1. Reporter Gene Constructs Promoter Reporter Target Gene Sequence LacZ (experimentally mutated) Luciferase ie; TAAT site, flanking sequence adjacent protein binding motifs Put reporter plasmid and a Hox gene expression plasmid into cell line Lyse cells, provide substrate Analyse colour/light output – do alterations to target sequence change output? - What changes to Hox gene alter output? Example: Luciferase Assay Examples of Mutations to Hox Expression Plasmid Alter sequence encoding 3rd helix (binding specificity) Alter Sequence encoding partner interacting domains Alter Sequence encoding NH-terminal activation domain Examples of Mutations to Target Promoter Sequence Deletion mutants to see WHICH TAAT sites are important Mutate TAAT site https://www.goldbio.com/articles/article/ a-deep-dive-into-the-luciferase-assay- Mutate TAAT flanking sequences what-it-is-how-it-works-and-more Mutate dimerizing partner DNA binding motif Dnase Footprinting Assay Add target DNA sequence and transcription factor (Hox) Incubate at physiological conditions Add DNase for a short while Enzyme will generate random lengths of digested DNA EXCEPT - where protected by Hox - https://www.chegg.com/homework-help/dnase- footprinting-experiment-either-template-nontemplate- st-chapter-5-problem-3aq-solution-9780073525327- exc Co-operativity binding of one ligand molecule to a protein influences the binding of subsequent the molecules to lig and same protein clustering - slightincreasare Steeper increase in gene activity With binding of one TF increased increasing IF concentration does not of affect the probability If molecules of Finding binding binding and to Others DNA Georgetti et al., 2010 Molecular cell 37(3):418-28 Gene Mobility Shift Assay (GMSA) binds when a protein a - DNA Fragment - mobility affected through get STEPS : 1. Preparation of DNA probe 2 Protein. binding 3 - Electrophores is. 4 Detection migrate Slower faster migrate Holden et al.,(2011) J. Pharmacol. Tox. Methods. 63(1): 7-14,  GMSA Cont’d Supershift (Ab, TF, Probe) TF dimer, Probe TF monomer, Probe Unincorporated Probe Modified from https://www.signosisinc.com/product/s mad-madh-emsa-kit-gs-0039 ”Knockout” Mice know steps/preparation https://en.wikipedia.org/wiki/Knockout_mouse “Knockout” Mice Summary 1. Hox binding is mediated by the 3rd helix of the homeodomain to the major groove - of DNA at a consensus site (TAAT) - 2. The N-terminal arm of Hox reaches out and makes contact with the minor groove - - 3. The pentapeptide motif binds partner transcription factors such as the homeobox gene exd/Pbx or MEIS 4. Amino acid sequence flanking the DNA binding domain matters - 5. The binding motif on DNA is contextually accessible: Flanking sequence matters - Other DNA binding motifs, and their relative spacing matters - 6. The presence and amount of transcription factor matters to: Form homodimer Form heterodimers - 7. Concentrations of Hox protein matter – co-operativity - - Problems Mutant Phenotypes Not Always as Predicted: Expect transformations at anterior border of expression 1. Transformations do not always occur at Hox expression boundary 2. Next gene does not seem to specify its usual somite/vertebral identity, but to specify generically one somite more posterior (order of somites proceeds smoothly without interruption) 3. Mutant phenotypes sometimes cover broad regions, and don’t perturb just at the boundary 4. Mutant phenotypes sometimes present in re-iterative manner 5. Deletion of entire cluster has surprisingly little effect None of these exceptions is consistent with the notion of the Hox code specifying precise spatial mapping identity (ie a particular Hox gene specifies somite contributing to vertebra T4 etc.).  Hox genes work w other signaling pathways. Hox genes redundancy show - one gene , others might compensate mutated Anomalies Hox is not static gene expression where new positional - info (posteriorizing) Hox genes normally exhibit is installed in body changes sharp anterior boundaries of plan imp expression for correct iden. of body segments along anterior posterior axis - According to the Hox code > - boundaries coincide model, this is where new body seg. wo transitions between phenotypes don't positional (posteriorizing) Cervical always thoracic) to map directly information should be to Hox normal gene installed and phenotypes boundaries should change align with borders of Irrespective of normal Hox body transitions boundary, phenotpyes map - to borders of body transition - (cervical to thoracic, thoracic - to lumbar etc.) - Some Explanations 1. Transformations do not always occur at Hox expression boundary Explanations? Somite re-segmentation could fuzzy the phenotype Functional redundancy of Hox genes could compensate and delay manifestation of phenotype 2. Next gene does not seem to specify its usual somite/vertebral identity, but to specify generically one somite more posterior (order of somites proceeds smoothly without interruption) Explanation? In the leap to “catch up” and cover for the missing previous posterior cue, some of the morphological attributes are smoothened The rest of the anomalies are incompatible with a Hox code – position model, and especially cannot explain why, when the entire Hox C cluster is deleted, there is little effect on spine. Problems with the neo-cassette Neo-cassette inserted into disrupt structure/function of gene/surrounding normal genome target a - the gene Homologous KO targeting vector introduces and artifact separate regulatory disrupt elements coordinated from genes expression > - Neo resistance selection marker acts as a genetic insulator – activity of adjacent - genes no longer coordinated. This is a product of: unexpected phenotypes Genes sharing regulatory elements diff interpret effects of gene knockout to Chromatin domain integrity being violated and not behaving properly (architecture is abnormal, epigenetic factors altered) - Timing of gene activation is thrown off, and since there is normally cross talk… this too is disrupted potentially could effect gene mask the of the knockout Solution? after knockout neo-cassette gene Cre-Lox removal of selectable marker remove 1. LOXP sites - insert LoxP sites) DNA sea recognized by Cre-recombinase enzyme) FLANK NEO-CASSETTE & Cre recombinase + LoxP sites - 2. Cre-recombinase expression excise specific DNA fragment - or delete a 3. Recombination cre-recombinase LoxP Sites recog - - excises DNA sequence between them  domains of adjacent Periodic pattern-expression Hox in a Insert Selectable Marker genes overlap ↓ manner regular select for cells that A. 1. Selectable marker insertion 1 2 3 4 5 6 7 8 9 10 11 12 13 have undergone 2. LoxP sites gene targeting and recombinase Cre Hoxa ↓ 3. Excision of Hoxb selectable Hoxc loss of periodicity disrupt normal marker. Marker-Free 4 Hoxd organization Cells Consensus Periodicity: d B. disrupt periodicity z Hoxa Hoxb Hoxc Hoxd new boundry formed C. Consensus Periodicity: Hoxa Hoxb Hoxc Hoxd loss in complete periodicity Consensus Periodicity: Conclusions Specific Hox crucial as gene might the not be TIMING - as of its activation Hox clusters are operating to posteriorize in a generic manner, not positionally codified TIMING of gene activation is critical, not WHICH specific gene is active Example from other Studies Engrailed – a homeobox repressor transcription factor Causes big neural problems when mutated in fly When knocked out in mouse, almost no discernable effect (subtle brain defect and learning disability) There is a second Engrailed gene in mouse En1 and En2 turn on at slightly different times during brain development En1 and En2 share only 55% amino acid similarity If En2 is expressed in place of En1 in a En1-/- background – phenotype is rescued in of Ent if expressed End is place in mouse Enl En1 Promoter En2 Open Reading Frame lacking normal e phenotype rescued of TIMING expression is more important than gene identity