Histone Code and Chromatin Structure

The Histone Code

• Histone modifications on a given nucleosome near a gene's control region affects the gene's transcription efficiency.
• This code is epigenetic and does not affect the base sequence of DNA.

Histone Modification and Transcription

• Activators in the IFN-β enhanceosome can recruit a HAT (GCN5).
• HAT acetylates some Lys on H3 and H4 in a nucleosome at the promoter.
• Protein kinase phosphorylates Ser on H3.
• This permits acetylation of another Lys on H3.
• Remodeling allows TFIID to bind acetylated Lys in the nucleosomes through the dual bromodomain in TAFII250.
• This "paves the way" for transcription to begin.

Chromatin States

• Euchromatin is relatively extended, open, and potentially active.
• Heterochromatin is very condensed, making DNA inaccessible.
• Heterochromatin can silence gene activity up to 3kb away.
• It is found at telomeres (ends) of chromosomes.
• Silencing of genes near the telomere is called the "telomere position effect."

Histone Methylation

• Certain proteins involved in forming heterochromatin have conserved regions called "chromodomains."
• Histone methyltransferase and associated protein HP1 are examples.
• HP1 binds to H3 when lysine 9 of H3 is methylated.
• This binding recruits histone methyltransferase to methylate lysine 9 on a neighboring nucleosome, propagating the silenced chromatin region.
• Methylation can also activate transcription (H3K4).
• The effect of histone modification depends on the identity, not just the nature, of the modification.

Interactions Among Modifications of H3

• Acetylation of K14 is needed for gene activation (INF-β).
• K14 acetylation depends on S10 phosphorylation.
• Phosphorylation of S10 is inhibited by methylation of K9.
• Phosphorylation of S10 and acetylation of K14 block methylation of K9.
• Acetylation of K9 prevents its methylation.

Signal at the Branch

• Along with consensus sequences at the 5' and 3' ends of nuclear introns, branchpoint consensus sequences also occur.
• Yeast sequence invariant is UACUAAC.
• Higher eukaryote consensus sequence is more variable.
• Branched nucleotide is final A in the sequence.
• The branchpoint consensus dictates the 3’ intron-exon boundary, typically ~35 nt downstream of the branch.

Spliceosomes

• Splicing intermediates are not free in the nucleus but bound to 40S particles called spliceosomes (60S in mammalian).
• Labeled yeast pre-mRNA incubated with yeast splicing extract.
• The mixture was subjected to glycerol gradient centrifugation,
• Radioactivity was determined within each gradient fraction.
• RNAs analysis from the fractions showed splicing intermediates and spliced out introns.
• Wild-type pre-mRNA and Mutant pre-mRNA have an A to C mutation at branchpoint.
• Wild-type pre-mRNA associated with the 40S complex.
• Mutant associates much less.

snRNPs

• Small nuclear RNAs coupled to proteins are abbreviated as snRNPs, small nuclear ribonuclear proteins.
• The snRNAs (small nuclear RNAs) can be resolved on a gel:
• U1, U2, U4, U5, and U6
• All 5 snRNAs join the spliceosome to play crucial roles in splicing.

Recognition of pre-mRNA

• U1 recognizes the 5' splice site first, then is replaced by U6.
• U2 recognizes the branchpoint.
• U2 Associated Factor binds to the 3' splice site.
• U5 binds to the 5' and 3' splice sites after recognition by other factors.

Splice Site Selection

• snRNPs do not have enough specificity and affinity to bind exclusively and tightly at exon-intron boundaries
• Additional splicing factors are needed to help snRNPs bind.
• Some splicing factors are needed to bridge across introns and exons and so define these RNA elements.

Commitment

• Commitment to splice at a given site is determined by RNA-binding protein
• This protein binds to splicing substrate and recruits other spliceosomal components, the first often being U1
• SR proteins (serine and arginine rich) SC35 and SF2/ASF commit splicing on human β-globin pre-mRNA and HIV tat pre-mRNA, respectively.
• In the presence of excess 5' splice site, splicing is inhibited.
• Preincubation with the SR protein SC35 can commit the pre-mRNA to splicing even with a competitor.

Bridging Proteins and Commitment

• Yeast commitment complex has a branchpoint bridging protein (BBP) that binds to:
  • U1 snRNP protein (yPrp40p) at the 5' end of the intron.
  • Mud2p near the 3' end of the intron.
  • RNA near the 3' end of the intron.
• It bridges the intron and could play a role defining intron prior to splicing.
• Mammalian BBP is SF1, may serve the same bridging function.

Yeast Two-Hybrid Assay

• The yeast two-hybrid screen takes advantage that the DNA binding domain (BD) and the activation domain (AD) of a transcriptional activator can be separated.
• To test if two proteins can make a physical interaction in vivo, each protein can be made as a fusion protein to either the BD or AD.
• If the two proteins interact, they will bring an AD in close proximity to a reporter gene engineered with an upstream binding site for the BD.
• To screen for an interaction, a protein of interest (the bait) is fused to the DB and a library of plasmids expressing many proteins (for example, a yeast cDNA library) is fused to an AD (the prey).
• Plasmids encoding fusion proteins that can interact with the bait will result in activation of the reporter gene and can be subsequently identified and characterized.

BioID

• It is a method that maps protein-protein interactions.

Alternative Splicing

• In humans, 60% of eukaryotic pre-mRNAs can be spliced in more than one way.
• This results in mRNAs that will encode different proteins.
• Transcripts of many eukaryotic genes are subject to alternative splicing.
• This splicing can have profound effects on the protein products of a gene.
• It causes a difference between secreted or membrane-bound protein.
• Activity and inactivity.
• Products of 3 genes in sex determination pathway of the fruit fly are subject to alternative splicing.

Alternative Splicing for Females

• Female-specific splicing of Sxl transcript results in protein that reinforces female-specific splicing of Sxl transcript and tra transcript, leading to active tra product.
• The active tra product, along with active tra-2, causes female-specific splicing of dsx transcript.
• dsx product inactivates male-specific genes and results in female development.

Alternative Splicing for Males

• Male-specific splicing of Sxl transcript gives an inactive product because of an exon with a stop codon.
• This permits default (male-specific) splicing of tra transcripts, which again leads to an inactive product because of a stop codon.
• With no tra product, dsx transcripts are spliced according to the default pattern, yielding a product that inactivates female-specific genes, therefore leading to male development.

Alternative Splicing in Stem Cells

• Sequencing of embryonic stem cells reveals an exon that is over-represented in these cells.
• Exon 18b (FOXP1-ES) is found in undifferentiated cells but is lost (changes to FOXP1) as cells differentiate into neural progenitor cells (NPC day 10).
• It is absent from many terminally differentiated cells (RT-PCR).
• Inclusion of exon 18b to make FOXP1-ES changes the amino acids of the ORF in the Forkhead DNA-binding domain.
• Flipping between exon 18 and exon 18b changes the DNA sequence bound by the transcription factor
• This is confirmed by gel mobility shift assay where FOXP1 and FOXP1-ES prefer different DNA-binding sequences.
• Depletion of the FOXP1 protein results in the downregulation of genes involved in organ and anatomical structure morphogenesis (development).
• Depletion of FOXP1-ES results in the downregulation of genes in embryonic development/gastrulation, and the upregulation of genes involved in organ development and differentiation.
• FOXP1-ES activates pluripotency genes and turns off differentiation genes.
• FOXP1 turns on differentiation genes

Dscam gene

• Dscam (Down syndrome cell adhesion molecule) is a conserved gene involved in neuronal development.
• It has 12, 48, 33 and 2 alternative exons for exons 4, 6, 9 and 17, respectively.
• The pre-mRNA transcript for the Drosophila gene Dscam has the potential to generate > 38,000 versions of Dscam (there are only ~ 14,000 genes in the whole Drosophila genome).

Eukaryotic rRNA and tRNA Processing

• Like mRNAs, rRNAs and tRNAs must be processed.
• That is, pieces of the originally transcribed RNA must be removed before the RNA reaches the mature form.
• Unlike mRNAs, rRNAs and tRNAs contain extra RNA at the 5' and 3' ends that must be removed.
• In eukaryotes, the ribosomal RNAs that will eventually make up the ribosome are initially transcribed by pol I as a long 45S rRNA precursor.
• First the 5’ spacer is removed, then the 41S precursor is cleaved into the 20S and 32S precursors. The 32S is cut into the mature 5.8S and 28S rRNAs, and the 20S is trimmed into the 18S.
• The 5.8S and 28S rRNAs base pair in the mature ribosome.

Ribosomal RNA Processing

• rRNA genes of both eukaryotes and bacteria are transcribed as larger precursors to yield mature-sized rRNAs.
• Several different rRNA molecules are embedded in a long precursor and must be cut out.
• In eukaryotes, many copies of the DNA encoding the rRNA genes are clustered together, separated by NTSs (non-transcribed spacers).
• The ribosomal RNAs are the most highly transcribed RNAs in the cell.
• The gap between the rRNA genes and NTSs is clearly visible by EM.
• The rRNA genes are concentrated to a distinct, electron-dense region of ribosome assembly in the nucleus, called the nucleolus.

rRNA Processing Order and Specificity

• The order and specificity of rRNA processing is orchestrated by small nucleolar ribonucleoproteins, or snoRNPs.
• snoRNPs contain small nucleolar RNAs (snoRNAs).
• snoRNPs direct and order splicing in the spliceosome.
• They interact with the rRNA precursor and direct where processing will occur and assist the ribosomal RNA in reaching the correct conformation to make a functional ribosome.
• This means they have RNA chaperone function.

Processing Bacterial RNA

• Like in eukaryotes, the bacterial rRNA is primarily transcribed as a long precursor.
• RNase III is important in the cleavage of the precursor into mature forms.
• The sequences flanking the 23S rRNA gene are predicted to create an extended hairpin, which is the recognition site for this enzyme.
• The cleavage sites on either side of the hairpin are offset by 2 nucleotides

tRNA Processing

• Transfer RNAs are made in all cells as overly long precursors (pre-tRNAs).
• These must be processed by removing RNA at both ends.
• Nuclei of eukaryotes contain precursors of a single tRNA.
• In bacteria, the precursor may contain one or more tRNA.
• Like for bacterial rRNA processing, the enzyme that first cleaves this into individual tRNA precursors is RNase III.

RNase P Action

• The first step in tRNA processing is the removal of the extra RNA at the 5' end of the pre-tRNA, called the leader.
• This is performed in both prokaryotes and eukaryotes by an enzyme called RNase P.
• This yields the correct, mature 5' end of the pre-tRNA.
• The RNAse P enzyme has two components: one protein and one RNA.
• The catalytic component of RNAse P is RNA.
• Catalytic RNA was discovered by Sid Altman (a Nobel prize winner along with Tom Cech) in 1989.