Lecture 23 Transcription, chromatin, RNA processingKey learning goals:

• Be able to explain the di!erence between heterochromatin and euchromatin.

• Understand that chromatin remodeling factors control nucleosome placement.

• Understand that chromatin structure and gene expression are controlled by covalent modifications of DNA and histones.

• Be able to explain the major functions of DNA methylation in eukaryotes. Understand that these functions di!er from the functions of DNA methylation in bacteria.

• Understand the functions of histone methylation and histone acetylation.

• Explain how transcriptional activators can regulate DNA and histone covalent modifications

• Know how the mRNA 5’ cap is added, and its structure.• Know how the mRNA poly-A tail is added. • Understand key features of RNA splicing reactions• Understand how mRNA modifications are coupled to transcription

D. Fawce", The Cell

Heterochromatin and euchromatin: regions of repressed and activated transcription

Histone remodeling complexes:

Motors that use energy of ATP hydrolysis to shove nucleosomes back and forth on DNA. These enzymes resemble helicases, but lack the ability to directly unwind DNA

Transcription initiation is regulated by DNA accessibility, by changing chromatin structure

(promoter sequence hidden)

(promoter sequence exposed)

Writer enzymes add marks to chromatin.

eraser enzymes remove these marks.

Note: marks can be placed on histones or DNA!

To produce a biological readout, reader proteins specifically bind to the histone modification.

Liu et al., 2010 Ann Rev Plant Biol 61:395

The structure and activity of chromatin is controlled by post-translational modifications

Chromatin condensationChromatin condensationCrhomatin de-condensationTransctiption activator binding…etc.

Some enzymes that write and erase chromatin marks

DNA methyltransferases (DNMTs) ☜write

histone acetyltransferases (HATs) ☜write

histone deacetylases (HDACs) ☜erase

histone methyltransferases (HMTs) ☜write

lysine demethylases (LSDs) ☜erase

Also: Histone kinases, histone ubiquitin ligases...

Covalent modification of DNA:• methylation (Cytosine C-5 in CpG dinucleotides)

• In mammals, 60-90% of all CpGs are methylated!

The structure and activity of chromatin is controlled by post-translational modifications

• enzymes: DNA methyltransferases• substrates: DNA (CpG) and SAM

The groups needed to form a Watson-Crick base pair with guanosine are not modified by this type of methylation

Covalent modification of histones:

• phosphorylation (Ser, Thr)• acetylation (Lys)• methylation (Arg, Lys)• ubiquitylation

note that phosphorylation adds a negative charge, while acetylation and methylation neutralize positive charges.

The structure and activity of chromatin is controlled by post-translational modifications

N-terminal tails of histone proteins are major targets of regulatory modification

VVP 3/e fig. 28-22

Methylation of histone Lys and Arg residuesby Histone Methyl Transferases (HMTs)

Liu et al., 2010 Ann Rev Plant Biol 61:395

You don’t need to know detailed names of the enzymes or methylation products on this slide — I just want you to get an idea of the potential complexity of this system.

As with DNA methyltransferases, the methyl donor substrate is SAM (S-adenosylmethionine)

Mosammaparast and Shi, Annu. Rev. Biochem. 2010. 79:155–79

Compliated pa!erns of histone tail methylation are associated with di"erent pa!erns of gene activity

We will focus on methylation marks that repress or silence genes.

H3-K27di-and tri-methylation

In general: Active genes have heavily acetylated histones

“Primed” genes (inactive genes in an activatable state) have less-heavily acetylated histones

Heterochromatin: Silenced genes are not very heavily acetylated, but are heavily methylated on DNA and on histones

Modified from Feinberg & Tycko, Nature Reviews Cancer 4, 143-153. Micrograph from D. Fawce", The Cell.

Methylation and maintenance of silenced heterochromatinby writers, erasers, and readers

Modified from Feinberg & Tycko, Nature Reviews Cancer 4, 143-153.

Methyl-C binding proteins (MBD) can be loaded onto methylated DNA through their interactions with both HDACs and HMTs.

Histone (H3) modifications include lysine (K) acetylation (Ac) and lysine methylation (Me). Lysines at other positions are also modified.

The HP1 protein recognizes MeK9 and, as this protein also binds the histone methyltransferase (HMT), heterochromatin can spread.

Histone deacetylases (HDAC) deacetylate lysine residues as a prerequisite for their subsequent methylation.

DNA methyltransferases (DNMT) participate in multiprotein complexes that contain HDACs and HMTs.

Methylation and maintenance of silenced heterochromatinby writers, erasers, and readers

Methylation begets more methylation.This allows silencing to be self-reinforcing

If the chromatin state controls transcription, howdo DNA-binding proteins control the chromatin state?

activator

enhancersequence

(promoter sequence hidden)

HAT

TATARNAP II

GTFs

enhancersequence

mediator complex

promoter sequence

activator

Transcription initiation is regulated by DNA accessibility through chromatin structure

• Histone Acetyl Transferases (HATs) acetylate lysines, especially on the N-terminal tails of histones.

• Many di"erent proteins, including remodeling factors, bind to acetylated lysines and other modified residues on histones.

In addition to TBP, TFIID alsocontains coactivator proteins.

At least one of these,TAF1, is a HAT enzyme!

TBP

TATARNAP II

GTFs

promoter

HAT (TAF1) TBPTFIID:

activatorprotein

enhancersequence

(promoter sequence hidden)

HATs

TATARNAP II

GTFs

promoter

TFIID

mediator

enhancerenhancer

TATARNAP II

GTFs

TATARNAP II

Nascent RNA chain

RNAP IIC-terminal domain(CTD)

P

PP

RNAP II C-terminal domain (CTD) is a floppy, intrinsically disordered part of the RNAP II enzyme

Transcription elongation: as RNAP II clears the promoter, the RNAP II CTD gets hyper-phosphorylated

COOH

COOH

5´

Histones are acetylated, displaced, and repositioned during transcriptional elongation

Once the PIC is assembled, the C-terminal domain (CTD) of RNAP II is phosphorylated many times…

…allowing the Elongatorcomplex to bind, and…

Elongator contains a histone acetyltransferase that helps to displace nucleosomes!

nucleosome-free gap of !200 bp over the promoter,flanked by two well-positioned nucleosomes. Thenucleosome-free regions include known DHSs and occu-pied transcription factor-binding sites. The nucleosomesflanking the nucleosome-free region appear to bedeacetylated and enriched in the histone variant H2A.Z(Liu et al. 2005; Raisner et al. 2005) Moreover, H2A.Zplays a role in the activation of gene transcription, yetappears to be concentrated on the promoters of genesthat are silent (Santisteban et al. 2000; Guillemette et al.2005; Li et al. 2005; Raisner et al. 2005; Zhang et al.2005). Thus, these data suggest that silent but inducibleyeast genes are poised with a nucleosome-free DHS atthe promoter flanked by two H2A.Z-containing nucleo-somes (Fig. 1A).

A somewhat different picture emerges from analysis ofDNA sequences in anti-histone chromatin immunopre-cipitates using lower-resolution yeast genome-wide mi-croarrays. These studies, with microarrays that includedamplified intergenic regions or the entire yeast genomeat 1-kb resolution, revealed an apparent depletion ofnucleosomes at active regulatory elements (Bernstein etal. 2004; Lee et al. 2004). They demonstrated an inversecorrelation between nucleosome occupancy at upstreamregulatory elements, as measured by the presence of his-tones, and rates of transcription of the correspondingORF (Bernstein et al. 2004; Lee et al. 2004). Interestingly,alteration of transcription programs by treatments suchas a change in carbon source resulted in newly repressedgenes gaining nucleosomes, while newly activated geneslost nucleosomes (Lee et al. 2004). As discussed in moredetail below, these studies also showed that the mosthighly transcribed genes are often partially depleted ofnucleosomes in coding regions (Lee et al. 2004).

The above observations appear, at first glance, to be in-consistent. Nuclease mapping studies (Ercan and Simp-son 2004) and high-resolution chromatin immunopre-cipitation studies (Yuan et al. 2005) both indicate thatmost promoters have hypersensitive sites regardless ofactivity, whereas genome-wide studies suggest that thenucleosome density of promoters is inversely propor-tional to transcription activity (Bernstein et al. 2004; Leeet al. 2004). These apparent differences might be recon-ciled, however, if additional nucleosomes are lost upongene induction (see Fig. 1B). Indeed, there are many ex-amples of such a phenomenon in the literature. Perhapsthe yeast PHO5 gene, studied extensively by Horz andcolleagues, is the best-characterized example of an in-ducible promoter in which nucleosomes are lost uponinduction (for review, see Svaren and Horz 1997). Strik-ingly, this group observed that histones at the promoterwere acetylated by GCN5 (the catalytic subunit ofSAGA and other acetyltransferase complexes) and sub-sequently lost contact with the promoter DNA (Reinkeand Horz 2003). In a related paper, Boeger et al. (2003)reported that these nucleosomes were actually unfoldedsuch that the histone octamer was no longer wrappedwith DNA. Notably, these nucleosomes were still lostwhen the PHO5 promoter was placed in small chromo-some circles, suggesting that they were lost in trans

rather than by sliding along the DNA in cis (Boeger et al.2003, 2004; Korber et al. 2004, 2006).

Figure 1. Model of nucleosome dynamics during transcriptionof a yeast gene. (A) Silent but inducible yeast genes contain anucleosome-free gap at the upstream activation sequence (UAS)that may be occupied by one or more sequence-specific bindingfactors (green ovals) flanked by two H2A.Z-containing nucleo-somes. (B) Upon activation, additional DNA-binding activators(blue ovals) are bound to the UAS that then recruit SAGA andSWI/SNF complexes. SAGA acetylates promoter nucleosomes,which facilitates the displacement of the H2A.Z-containingnucleosomes and perhaps additional nucleosomes by SWI/SNF.The histones are transferred onto the Asf1 and Nap1 histonechaperones. (C) The resulting nucleosome-free region is thenavailable for the formation of a preinitiation transcription com-plex including RNA polymerase II. (D) During transcription,elongation acetyltransferase complexes associated with the elon-gating polymerase acetylate nucleosomes in front of the polymer-ase. This facilitates their partial or complete disassembly, trans-ferring the histones onto the Spt6 and FACT histone chaperones.Spt6 and FACT reassemble nucleosomes behind the polymerase,and histone H3 is methylated by the polymerase-bound Set2methyltransferase. The Eaf3 subunit of the Rpd3S histone deacety-lase complex recognizes the methylated histone H3 and deacety-lates those nucleosomes to restore their previous stability. Thereassembled nucleosomes behind polymerase may also containnew histones from the soluble cellular pool.

Workman

2010 GENES & DEVELOPMENT

Cold Spring Harbor Laboratory Press on February 21, 2008 - Published by www.genesdev.orgDownloaded from

mediator

As it moves, Pol II leaves a “trail of breadcrumbs” as it transcribes the gene!

• 5´cap addition• 3´polyadenylation• splicing, to remove introns

mRNA processing:

This stu" happens during elongation!

–poly-A–3´5´cap–5´ UTR 3´ UTR

Coding sequence:translation start translation stop

introns spliced out

The 5´end of the nascent mRNA is CAPPED by 7-methylguanosine

Capping Enzyme does this in three steps:

1. Hydrolysis removes 5´ γ-Pi from pre-mRNA.

2. Guanylation of pre-mRNA through 5´ to 5´ triphosphate bridge (PPi released).

3. Methylation of the guanosine base at position N7. Methyl donor is SAM (S-andenosylmethionine).

Capping occurs during elongation. Capping enzyme binds to the RNAP II phosphorylated CTD

Eukaryotes only! — bacterial mRNA has 5´-PPP

The 3´end of the nascent mRNA is polyadenylated

Poly-A tails (3´-An) contribute to mRNA stability in the cytoplasm.

3´-An are added by non-templated addition by the poly-A polymerase (PAP).

Polyadenylation is coupled to transcription.

The polyadenylation machinery associates with:

1. The phosphorylated RNAP II CTD;

2. AAUAAA sequence in the mRNA. This sequence marks the approximate position of 3´-An addition.

Most eukaryotic mRNA transcripts are spliced

Introns are removed. Exons remain.

alpha-tropomysin pre-mRNA (encodes a structural protein)

Some transcripts exhibit tissue-specific splicing

exonskipping

exonskipping

Lariat Larry (dude really does call himself that)

Two nt’s at the beginning and end of an intron are invariant

Most introns are removed through the lariat mechanism

Lariat mechanism — most common way mRNAs are spliced

mRNA splicing is carried out by the SPLICEOSOME, a snRNP (small nuclear ribonucleoprotein – say “snurp”)

The adjective “small” is misleading, though. The RNA components are small compared to some other RNAs, but this is an enormous (45S) complex, one of the biggest in the cell!

Joan Steitz

This is the U7-snRNP, which controlsprocessing of the 3´end of the mRNAs

encoding histone proteins.

1

2

3

4

Group I introns are autocatalytic: they splice themselves!Certain eukaryotic ribosomal RNAs do this. This was the first example of RNA catalysis. It was u!erly unexpected! Many, many other examples have since been discovered.

h"p://www.annualreviews.org/doi/pdf/10.1146/annurev.bi.59.070190.002551