Differential Gene Expression and Cell Differentiation

Differential Gene Expression and Cell Differentiation

• Embryonic development involves a single cell differentiating into many cell types
• Somatic cell nuclei each contain the same chromosomes and genes
• Genomic equivalence stipulates all body cells contain the same genes

Selective Gene Activation

• Differential gene expression makes cells different based on the combination of active genes
• Selective protein production promotes cellular diversity
• Gene expression differences govern maturation toward distinct cell types during zygote division
• Regulatory mechanisms modulating differential gene expression target DNA access, RNA production/processing, protein synthesis/modification include:
  • Specific transcription factors binding gene promoters
  • Histone modification to alter chromatin accessibility
  • RNA degradation and alternative splicing,
  • Translational controls
  • Post-translational protein modifications
  • Protein transport changes

Postulates of Differential Gene Expression

• Somatic cells contain the complete genome from the fertilized egg
• Unused genes are not destroyed or mutated, retaining expression potential
• Only a small genome percentage expresses itself in each cell
• RNA synthesized is unique to the certain cell type

Gene Expression Regulation Levels

• Gene expression has four regulation levels
  • Differential gene transcription regulating transcribed nuclear genes into nuclear RNA
  • Selective nuclear RNA processing regulating transcribed RNAs that enter the cytoplasm/become messenger RNAs
  • Selective messenger RNA translation regulating mRNAs translated into proteins
  • Differential protein modification regulating proteins to remain and function in a cell
• Some genes, like globin protein subunits of hemoglobin, regulate at all levels

Genomic Equivalence Evidence

• Polytene chromosomes in Drosophila larvae exhibit no structural differences between cells
• However, chromosome regions "puff up" during RNA production
• Nucleic acid in situ hybridization studies confirmed these findings
• Odd-skipped gene mRNA indicates a segmented pattern in the Drosophila embryo
• The mouse homolog, odd-skipped related 1, uniquely expresses itself in branchial arches, limb buds, and the heart

Cloning and Nuclear Equivalence

• Nuclear transplant experiments demonstrate somatic cell nuclei of origin can direct an organism's entire development
• Dolly the sheep was cloned in 1997 using a mammary gland cell nucleus
• Cells were kept in the G1 phase, then fused with enucleated oocytes using electric pulses, activating development
• DNA analysis verified that Dolly's cells originated from the donor nucleus strain
• Cloning of adult mammals has since occurred in other animals
• Nuclei of vertebrate adult somatic cells contain the required development genes
• Somatic cell nuclei are equivalent

DNA and Chromatin

• Eukaryotic (but not prokaryotic) genes are contained in the DNA and chromatin complex
• Histones form the protein component of chromatin, particularly the nucleosome
• An octamer creates the nucleosome of histone proteins (two molecules each of H2A, H2B, H3, and H4) wrapped with two DNA loops (approx. 147 base pairs)
• Histone H1 binds linker DNA (60-80 base pairs) between the nucleosomes
• DNA has multiple contacts with histones to enable packaging into the nucleus

Nucleosomes

• Solenoids tightly wind the nucleosomes, stabilized by histone H1
• H1-dependent nucleosome conformation inhibits somatic cell gene transcription
• Tightly packed chromatin regions are called heterochromatin
• Loosely packed areas are called euchromatin
• Regulating how tightly packed an area of chromatin is modulates gene accessibility for transcription

Histones as Gene Expression Gatekeepers

• Modifying histone "tails" of H3 and H4 controls repression and activation
• Histone acetylation (adding negatively charged acetyl groups) loosens the histones and activates transcription
• Histone acetyltransferases add acetyl groups, destabilizing nucleosomes and making them euchromatic
• Histone deacetylases remove acetyl groups, stabilizing nucleosomes to prevent transcription (heterochromatic)

Histone Methylation

• Methylation can activate/repress transcription
• Acetylation, along with three methyl groups on lysine at position four of H3 (H3K4me3), usually means active transcription
• Lack of acetylation and methylation on the lysine in the ninth position of H3 (H3K9) means repressed chromatin

Memory of Methylation

• Histone modifications recruit proteins from the Trithorax and Polycomb families, retaining the transcriptional state from generation to generation
• Trithorax proteins keep active genes active
• Polycomb proteins maintain a repressed state through condensed nucleosomes

Polycomb Proteins

• Polycomb proteins repress sequentially
  • First set methylates lysines H3K27 and H3K9 to repress a gene
  • Second set binds methylated tails of histone 3, keeps methylation active, methylates adjacent nucleosomes, forms tightly packed repressive complexes

Trithorax Proteins

• Act to counter the Polycomb proteins effects
• Modify/alter nucleosome positions, allowing transcription factors to bind DNA
• Trimethylated H3K4 lysine is kept from from demethylating into a dimethylated, repressed state

Anatomy of Eukaryotic genes

• Modulating gene access via histone methylation affects expressions
• Regulated directly through methylation targeting DNA
• Non-colinear with their peptide products
• Eukaryotic mRNA comes from noncontiguous regions on the chromosome
• The single nucleic-acid strand

Exons and Introns

• Exons : DNA regions that encode parts of a protein
• Introns: Intervening sequences with no information for coding
• 5' region: where RNA polymerase II binds
• Transcription initiation site: ACATTTG for human β-globin
  • the "cap sequence" where a modified nucleotide "cap" is added soon after transcription
• 5' untranslated region (5' UTR; leader sequence)
  • 50 base pairs
  • Determines translation rate
• Translation initiation site: ATG becomes AUG in mRNA
  • Located 50 base pairs after the transcription initiation site in the human β-globin gene
• Protein-encoding portion of the first exon
  • 90 base pairs for amino acids 1–30 of human β-globin protein
• An intron
  • 130 base pairs
  • Non-coding structure enables RNA processing and exit from nucleus
• Exon containing 222 base pairs
  • Amino acids 31-104 coded for
• Large Intron
  • 850 basepairs, no coding sequence
• Exon
  • 126 base pairs coding for amino acids 105-146
• Translation Termination Codon
  • TAA becomes UAA
  • signals that the ribosome dissociate and for protein release
• 3' untranslated region (3' UTR)
  • Includes polyadenylation sequence AATAAA
  • tail of 200-300 adenylate residues; stabilizes mRNA, allows nucleus exit, permits mRNA translation
• Transcription Termination Sequence
  • Continues beyond AATAAA site for ~1000 nucleotides before terminating

Nuclear RNA (nRNA)

• The original transcription product, containing the cap sequence, 5’ UTR, exons, introns and the 3’ UTR
• Undergoes ends modification before leaving the nucleus
• A methylated guanosine cap anchors the 5' end of the RNA; for mRNA binding to ribosome/translation
• A polyA tail of adenylate residues to the 3' end for stability/protection from exonucleases
• Introns are removed and exits spliced together

cis-Regulatory elements: the On/Off switches

• Promoters: Site with RNA polymerase II binds, starts transcription
• Most contain CpG islands
• Basal transcription factor proteins: saddle and recruit RNA polymerase II

Enhancers

• Recruit and stabilize RNA polymerase II
• Signals where/when to use a promoter, how much gene product to make
• Controls efficacy/rate of transcription from a specific promoter
• Transcription factors to bind specific promoters/enhancers/silencers
• Recruit enzymes that break up area nucleosomes