Drosophila Development Lecture Notes PDF

Lecture 10 – Transcriptional regulation continued A Eukaryotic Gene Control Region Consists of a Promoter Plus Many cis-Regulatory Sequences Transcription May be Regulated by Combinatorial Controls Segments in the embryo can be seen in the adult Drosophila embryos are segmented Early embryog...

Lecture 10 – Transcriptional regulation continued A Eukaryotic Gene Control Region Consists of a Promoter Plus Many cis-Regulatory Sequences Transcription May be Regulated by Combinatorial Controls Segments in the embryo can be seen in the adult Drosophila embryos are segmented Early embryogenesis in Drosophila Patterning genes are expressed in distinct parts of the embryo to specify cell fates Three Groups of Genes Control Drosophila Segmentation Along the A-P Axis A Hierarchy of Gene Regulatory Interactions Subdivides the Drosophila Embryo Identification of Patterning Genes in Drosophila Wieschaus and Nusslein-Volhard genetic screen for Drosophila embryonic lethal mutants identify mutants that arrest prior to embryo hatching and that appear normal in some parts of embryo but abnormal in other parts 3 general classes of mutant: gap genes – mutants lack entire regions of embryo pair rule genes – mutants lack parts of each segment segment polarity genes – part of each segment is replaced by a duplication (often in mirror image) of the other part of that segment In separate genetic screens they identified genes in which mutant females produce embryos lacking either posterior or anterior structures – eg. bcd Nobel prize in 1995 Egg-Polarity Genes Encode Macromolecules Deposited in the Egg to Organize the Axes of the Early Drosophila Embryo RNA in situ hybridization immunolocalization Bcd gradient determines pattern of hb and otd expression  Bicoid (Bcd) bcd mRNA is deposited in egg during oogenesis Bcd protein forms a gradient from anterior towards middle of egg Bcd is a homeodomain transcription factor that promotes transcription of genes required for anterior fates (Bcd can also bind RNA – can regulate translation) bcd gene is genetically required in the mother – maternal effect gene its targets – otd, hb (and others) are required in the embryo – zygotic genes Bcd gradient determines pattern of hb and otd expression 3 weak sites 3 weak sites + 3 strong sites Otd and hb genes have Bcd binding sites The two genes differ in number of binding sites and affinity for Bcd protein Gradient of Bcd protein determines expression of target genes Bcd protein gradient – decreasing levels towards posterior of embryo Hb, Giant (and Otd) – Bcd target genes Kruppel transcription is repressed by Hb (and by another repressor that is present at posterior) Giant is repressed by another transcription factor at anterior end (and is activated by another transcription factor near posterior). Bcd, Hb, Giant and Kruppel all regulate Even-skipped (Eve) Complex Genetic Switches That Regulate Drosophila Development Are Built Up from Smaller Molecules Even Skipped (Eve) – a pair-rule gene Complex Genetic Switches That Regulate Drosophila Development Are Built Up from Smaller Molecules Complex Genetic Switches That Regulate Drosophila Development Are Built Up from Smaller Molecules Eve stripe 2 expression depends on a specific regulatory sequence Complex Genetic Switches That Regulate Drosophila Development Are Built Up from Smaller Molecules Complex Genetic Switches That Regulate Drosophila Development Are Built Up from Smaller Molecules The Drosophila Eve Gene Is Regulated by Combinatorial Controls Giant represses eve transcription at the anterior of the embryo wild type giant mutant eve stripe 2 - lacZ Determining DNA sequences that a transcription factor binds to Identify DNA sequences that Giant binds to Chromatin IP (ChIP) Chromatin-IP followed by DNA sequencing (Chip-seq) DETERMINE SEQUENCE OF DNA Chromatin-IP followed by DNA sequencing (Chip-seq) To identify DNA sequences that a transcription factor binds to - add crosslinker (eg. formaldehyde) to cells.  proteins bind to nearby proteins (or DNA). - lyse cells - break DNA into small fragments - immunoprecipitate transcription factor - reverse cross links to elute DNA - next gen sequencing to identify DNA sequences Genome-wide Chromatin Immunoprecipitation Identifies Sites on the Genome Occupied by Transcription Regulators number of IP Giant sequence reads protein eve gene Giant ChIP reveals 3 sites upstream of eve gene and within eve stripe 2 enhancer (one of which is not shown here) Number of sequence reads indicates frequency of protein occupancy – relates to affinity (and protein concentration) The Drosophila Eve Gene Is Regulated by Combinatorial Controls * * * * site-directed mutagenesis of Giant binding sites eve stripe 2 – lacZ in wild type (control) eve stripe 2 – lacZ in giant mutant (for reference) eve stripe 2***- lacZ *** = mutations in all 3 giant binding sites in wild type The Drosophila Eve Gene Is Regulated by Combinatorial Controls Anterior to eve stripe 2 Eve stripe 2 Posterior to eve stripe 2 The Drosophila Eve Gene Is Regulated by Combinatorial Controls Eukaryotic Transcription Repressors Can Inhibit Transcription in Several Ways MOLECULAR GENETIC MECHANISMS THAT CREATE AND MAINTAIN SPECIALIZED CELL TYPES The Drosophila Eve Gene Is Regulated by Combinatorial Controls Eve stripe 2: binding sites for Bcd, Hb, Giant and Kruppel In order for Eve to be transcribed: Bcd and Hb must be bound Bcd sites are low-affinity  synergistic effect on RNA pol recruitment Giant and Kruppel binding can prevent binding of Bcd and Hb, or block their interaction with RNA Pol II Eve gene has binding sites for other transcriptional regulators (these activators or repressors are found in other parts of the egg)  Lecture 11 Post-transcriptional regulation of gene expression  Post-transcriptional regulation of gene expression alternative splicing regulation of nuclear export mRNA localization miRNA nonsense-mediated decay (lecture 12)  POST-TRANSCRIPTIONAL CONTROLS Alternative RNA Splicing Can Produce Different Forms of a Protein from the Same Gene Alternative RNA Splicing Can Produce Different Forms of a Protein from the Same Gene Nucleotide Sequences Signal Where Splicing Occurs RNA Splicing Removes Intron Sequences from Newly Transcribed Pre-mRNAs Spliceosome: U1 snRNP base pairs with 5’ splice jxn U2 snRNP base pairs with branch point Other splicosome subunits recruited Adenine in branch point “attacks” 5’ site  lariat formation and free 3’OH Alternative RNA Splicing Can Produce Different Forms of a Protein from the Same Gene Drosophila sex determination XX XY Sxlon Sxloff Traon Traoff female male Sxl inhibits binding of the Spliceosome to the proximal splice site U2-AF (U2)– part of Spliceosome that recognizes 3’ splice site Sex determination in Drosophila Transcription of Sxl in XX (female) embryos Sxl promotes alternative splicing of tra RNA Sxl binds to splice acceptor site for exon 2 on tra RNA  prevents Spliceosome interaction with this splice acceptor site Spliceosome interacts with alternative splice acceptor site (distal site) female tra mRNA is translated into Tra protein  determines female fate in absence of Sxl, default splicing of tra using proximal splice acceptor site  mRNA produces a truncated non- functional Tra protein  default male fate  Post-transcriptional control of gene expression by mRNA localization Some mRNAs Are Localized to Specific Regions of the Cytosol Sequences in the 3’ UTR determine localization Proteins bind these sequences and link the RNA to cytoskeleton Parts of a mature mRNA 5’ UTR Some mRNAs Are Localized to Specific Regions of the Cytosol Egg-Polarity Genes Encode Macromolecules Deposited in the Egg to Organize the Axes of the Early Drosophila Embryo bicoid mRNA is localized to the anterior of the egg bicoid 3’UTR has a sequence that is recognized by Staufen protein Staufen links bicoid mRNA to microtubules at the anterior of the egg When translated, Bicoid protein diffuses from anterior Mature Eukaryotic mRNAs have 5’ Cap and 3’ polyA tails pABP on 3’polyA tail interacts with proteins bound to 5’ Cap This structure protects mRNA from degradation  Post-transcriptional control of gene expression via changes in mRNA stability All mRNAs are subject to degradation. Major degradation pathway mediated by 5’ to 3’ exonucleases. 5’ cap protects mRNAs from degradation. Poly-A tail (with PABP bound) binds and protects 5’ cap. Deadenylation (loss of 3’ poly-A tail) leads to 5’ cap loss. poly-A tail shortening via deadenylase (a type of 3’ to 5’ exonuclease) gradually reduces poly-A tail length This leads to de-capping followed by degradation from 5’ end (5’ to 3’ exonucleases) Changes in mRNA Stability Can Regulate Gene Expression  REGULATION OF GENE EXPRESSION BY NONCODING RNAs Small Noncoding RNA Transcripts Regulate Many Animal and Plant Genes Through an RNA Interference pathway microRNAs –Ambros and Lee – 2024 Nobel Prize  Lin-14 and-Lin 4 Lin-14 transcription factor- mutants disrupt timing of cell divisions in C. elegans Lin-4 opposite phenotype double mutant (lin-14,lin-4) phenotype is same as lin-14 mutant  lin-4 is a negative regulator of lin-14 Ambros and Lee -1993 lin-4 encodes a non-coding micro-RNA that regulates lin-14 Lin-4 micro-RNA Isolation of 2 lin-4 RNAs lin-4L predicted to form a hairpin lin-4S – identical to part of longer RNA partial complementarity of lin-4S to several sites in 3’UTR of lin-14 Lee et al, 1993  Lin-14 and-Lin 4 Ambros and Lee -1993 lin-4 gene not predicted to encode a protein encodes a small RNA that is complementary to itself – forms a hairpin structure partial complementary to a sequence in 3’UTR of lin-14 lin-4 mutant  no effect on lin-14 mRNA levels, but increase in amount of lin-14 protein  lin-4 is a negative regulator of lin-14 translation Model: lin-4 RNA binds 3’UTR of lin-14 to block translation  RNA interference Discovered in 1998 Eukaryotic cells have machinery that processes double stranded RNA from viruses and uses this to defend against the virus Dicer – cuts dsRNA into 23nt pieces - siRNA RISC – removes one strand, uses other to identify and cut complementary ssRNA Small Noncoding RNA Transcripts Regulate Many Animal and Plant Genes Through RNA Interference from virus microRNA activity requires RNAi machinery 2001 –Dicer mutant has a similar phenotype to lin-4 (Dicer mutant also has other phenotypes)  lin-4 requires Dicer (and RISC) to block translation of lin-14  micro-RNA is processed by the same machinery that processes double stranded RNA for RNA interference  REGULATION OF GENE EXPRESSION BY microRNAs over 2,500 miRNAs are encoded in our genome regulate up to 1/3 of all coding genes transcribed via RNA polymerase II form hairpin structures recognized by RNAi machinery processed form of miRNA (guide RNA/RISC complex) targets one or more mRNAs  REGULATION OF GENE EXPRESSION BY microRNAs each miRNA has homology to sequences in 3’ UTR of specific target genes – eg. mir-21 targets several genes including PTEN (a gene that inhibits growth) miR-21 and PTEN 3’UTR can base pair – partial complementarity miRNAs Regulate mRNA Translation and Stability Drosha Dicer RISC guide strand in RISC target mRNA in P-body  miRNAs Regulate mRNA Translation and Stability transcription of miRNA cropping in nucleus – Drosha (endonuclease) export to cytoplasm Dicer (endonuclease)  cleavage to remove hairpin  generation of a 23 nucleotide dsRNA this is recognized by RISC RISC / Argonaute further mediates translational repression in P-body (and eventual degradation) Argonaute proteins  RISC Major component is an Argonaute protein Argonaute protein binds to a single strand from a dsRNA – the guide strand (and slices/degrades the other strand) RISC recognizes 5’ phosphate and 3’OH. RISC only binds RNA of ~23 nucleotides RISC complex seeks target mRNAs - mRNAs with sequence complementary (typically in 3’UTR) to miRNA If a perfect match  Argonaute slices (degrades) mRNA Most miRNA targets – imperfect match  translational repression in P-bodies Small Noncoding RNA Transcripts Regulate Many Animal and Plant Genes Through RNA Interference from virus perfect match partial match