Transcription and chromatin (Gene expression)

What do downstream promotors do?

A downstream promoter element (DPE) is a core promoter element. Like all core promoters, the DPE plays an important role in the initiation of gene transcription by RNA polymerase

What is a nucleosome? Is DNA left as nucleosomes?

A region of DNA wound around histone proteins A nucleosome comprises of 147bp of DNA wrapped around histones usually 2xH2A, 2xH2B, 2xH3 and 2xH4. A variable stretch of DNA separates nucleosomes which is stabilised by the histone H1 There is an average of 50bp between nucleosomes, but it varies a lot between regions of DNA The nucleosomes bind DNA non-specifically, as required by their function in general DNA packaging. There are, however, large DNA sequence preferences that govern nucleosome positioning. This is due primarily to the varying physical properties of different DNA sequences: For instance, adenine and thymine are more favourably compressed into the inner minor grooves. In the nucleus, the DNA NEVER exists as an extended chain of nucleosomes - it is instead further packed into thicker solenoid type structure, chromatin fibres and then to chromosomes.

What is the start codon?

AUG commonly. ATG The start codon is usually preceded by the untranslated 5' region.

What is an initiation element?

Also called an initiator motif Plays a similar role to TATA box It has the consensus sequence YYANWYY in humans Similarly to the TATA box, the Inr element facilitates the binding of transcription Factor II D(TAF). The Inr works by enhancing binding affinity and strengthening the promoter.

Draw out the key elements in the 5'UTR

Although these are contained with 5' UTR, which are critical for translation - these are also critical core promotors for transcription

What is nuclear localisation?

As we know chromosomes occupy specific regions of the nucleus. However, sequences that lay far apart In the nucleus, but are involved in regulatory interactions actually come together in the nucleus - this is known as gene kissing This can be seen by chromosome capture (3C/4C/Hi-C)

What are transcription factories?

Chromosomes occupy distinct territories within a nucleus When genes are being actively transcribed, the genes extend out into the interchromosomal space to allow gene expression They extend out into 'transcription factories'

What are the two types of chromatin remodelling?

Covalent histone modifying complexes ATP dependent chromatin remodelling complexes

Just look: another diagram

DNA hypermethylation, histone hypoacetylation and inactive chromatin repress transcription. In contrast, a transcriptionally active condition may encourage DNA hypomethylation, histone hyperacetylation and active chromatin. Also, a particular chromatin structure may be required for establishing DNA methylation. (B) DNA methylation and histone modification play key roles in transcriptional control. The figure shows transcriptional factors (TF), RNA polymerase (Pol II), general transcription factors (GTF), acetylated histone (Ac) and methylated cytosine (mC). Either HAT or HDAC recruited by TF and other DNA-binding proteins induce transcriptional activation or repression, respectively. Modifications (acetylation, phosphorylation, methylation) of histone tail domains are described in the text. Chromatin remodeling factors convert the chromatin to active (upper) and inactive (lower) states.

How are chromatin states maintained?

DNA methylation is not the sole trigger the formation of heterochromatin Different mechanisms to maintain chromatin states are used: HP1 protein Small RNA molecules Nuclear localization

What are GC Boxes?

GC box is a distinct pattern of nucleotides found in the promoter region of some eukaryotic genes upstream of the TATA box and approximately 110 bases upstream from the transcription initiation site. A large subclass of polymerase II promoters lacks both TATAA and CCAAT sequence motifs but contains multiple GC boxes.

What are transcription factors? What are their general structures? List some different types What do they help to form?

General transcription factors required for RNA pol II are detailed in the box below Each transcription factors are themselves made up of key proteins, such as TATA-box binding proteins. The complex that is required to start translation via RNA pol II is called the basal recognition apparatus. This consists of RNA pol II plus all of the associated transcription factors.

Describe the different steps in the formation of the basal transcription apparatus

Given a favourable chromatin environment, the basal transcription machinery will assemble on the DNA promotor This machinery assembles sequentially.

Distal regulatory elements are often clustered together. What are these called?

LCR regions or super regulators. As with the beta globin LCR on 11p. These regions contain enhancer and insulator regions (preventing more distal regulatory elements from interfering). However, many regulatory elements are more scattered. Its physiological levels of expression are directly proportional to transgene copy number in transgenic mice It is copy number dependent It is highly conserved in many species It was first discovered in relation to the beta globulin gene - but many more have now been discovered Without an LCR, genes would simply not be expressed

Describe the sense and antisense strand

One of the DNA strands is a template strand. The transcribed mRNA molecule is a copy of the complementary strand (the sense strand) The template strand is called the antisense strand.

What is chromatin remodelling?

The DNA can flip between Euchromatin and heterochromatin and VV when it requires This is called chromatin remodelling This solves the long unanswered question of how the DNA is read when it is tightly packed as chromosomes. Essentially, remodelling allows chromatin to relax its structure and reverse when inactive.

Are promotors or histone modifications more predictive of transcription?

The spacing of elements is also important Characteristic histone modifications can predict transcription start sites - as only 32% of promotors carry a TATA box

There are different types of histone which has different functions. Name some of these.

There are different types of histone. For example, H1 histone binds to the exterior of the nucleosome and pulls everything together. Histones are positively charged amino acids (Lysine and arginine). This makes them bond electrostatically to the negatively charged phosphate groups of nucleotides in DNA.

What are enhancers? How do they act?

These are clusters of cis acting short sequence elements that can enhance the transcription acticity of a specific gene. Unlike promotors, the position of enhancers are highly variable and can be miles away. These can also bind gene regulatory proteins, that when the DNA is coiled can interact with promotor sites.

How does acetylation of histones change chromatin structure?

Acetylation of histones H3 and H4 normally increases gene expression by promoting an open chromatin structure. Acetylation removes the positive charge on the histones, thereby decreasing the interaction of the N termini of histones with the negatively charged phosphate groups of DNA. As a consequence, the condensed chromatin is transformed into a more relaxed structure that is associated with greater levels of gene transcription.

What is a fractal structure? How does this relate to DNA?

After the beads on a string, the DNA coils into a fractal structure This means that the chromosome itself is self-similar at all scales, that is, chromosomes have structural features that are similar at many magnifications. Chromatin produces regularly spaced diffraction rings, that suggest a repeating structural unit occuring all along the chromatin axis.

What is a HP1 protein?

Heterochromatin protein 1 Small non-histone protein Highly conserved across evolution This probably mediates the spread of heterochromatin HP1 contains a chromodomain that enables it to bind to histones modified by the methylation of H3K9 methyltransferase Moreover, HP1 acts as a platform in which chromatin modifying proteins can be assembled

How are acetyl groups added and removed from histones?

Histone Acetyltransferases, also known as HATs, are a family of enzymes that acetylate the histone tails of the nucleosome. This, and other modifications, are expressed based on the varying states of the cellular environment. Histone deacetylases do the opposite.

What is the basic structure of chromatin?

If you look at a nucleosome on an electron micrograph you will see a "Beads on a string" appearance The basic repeat element of chromatin is the nucleosome, interconnected by sections of linker DNA Nucleosomes are organised into an octamer of proteins called a histone DNA wraps around a histone

What are CpG islands? What do they do?

In humans, about 70% of promoters located near the transcription start site of a gene (proximal promoters) contain a CpG island. Particularly in housekeeping genes. Distal promoters also frequently contain CpG islands In humans, DNA methylation occurs at the 5' position of the pyrimidine ring of the cytosine residues within CpG sites Silencing of a gene may be initiated by other mechanisms, but this is often followed by methylation of CpG sites in the promoter CpG island to cause the stable silencing of the gene.

List some different mechanisms by which transcription can be modulated, particularly at different time points and in different tissues

Lots of transcription factors are tissue specific and modulate transcription. Transcription factors are said to be trans-acting. Other DNA binding proteins such as enhancers and silencers located some way from the promotor also influence transcription but looping of the DNA brings them into close contact. These can lay upstream or downstream. Enhancers are cis-acting, but are often far away. These can also bind to regulatory proteins. (Silencers do the opposite) Developmental genes have a complex set of distal enhancers.

What are the three different types of polymerases?

Pol I Transcribes RNA genes that exist as tandem arrays on short arms of acrocentric chromosomes Pol II Transcribes all protein coding genes and many RNA coding genes including RNA, snRNA, miRNA Pol III Transcribe genes for transfer RNA and some smallRNA

What are core promoters? Give some examples

Promotors of pol II are highly heterogenous They often include TATA, GC and CCAAT boxes The core promoter is the minimal portion of the promoter required to properly initiate transcription. The core promoter is approximately -34 bp upstream from the Transcription start site.

Describe some of the events and key players in the start of transcription

RNA polymerase synthesizes the transcript. Transcription is highly regulated Pol II core promotors (TATA and CCAAT boxes as well as GC boxes) all play a key role in regulating transcription. The spacing of elements is also important. CpG island promotors are also more numerous than TATA boxes. For transcription to happen, you also require key transcription factors (and the activity of enhancers)

How do covalent histone modifying complexes work? What are the different ways histones can be modified?

Specific protein complexes, known as histone modifying complexes catalyse the addition or removal of various chemical elements on histones. These are post-translational modifications to the histone amino-terminal tails It alters chromatin structure, thereby changing DNA accessibility These can alter DNA histone contacts • This includes; ○ Acetylation (Done by histone acetyl transferase HAT and removed by HDAC) ○ Methylation (Done by HMT - Histone methyltransferase) ○ Phosphorylation ○ Ubiquitination (adding of ubiquitin groups) These extra groups do not bind to DNA themselves. They can be recruited by; ○ Histone modifications ○ Transcription factors ○ RNA ○ DNA damage Acetylation of histones H3 and H4 normally increases gene expression by promoting an open chromatin structure.

In relation to chromatin remodelling, how do UCOE's work?

Structure: methylation-free CpG island encompassing single or dual divergently transcribed promoters of ubiquitously expressed housekeeping genes. Possesses an extended DNA methylation-free region and associated active histone modification marks. Function: allows reproducible and stable expression of a linked transgene.

What are telomeres? What are the genomic structure of telomeres? Why do telomeres shorten? What enzyme replenishes telomeres?

Telomeres are specialised heterochromic DNA-protein complexes at the ends of linear eukaryotic chromosomes They are comprised of short tandem repeats that are well conserved During chromosome replication, the DNA polymerase enzyme that duplicates DNA cannot continue their duplication all the way to the end of a chromosome, so in each duplication the end of the chromosome is shortened (this is because the synthesis of Okazaki fragments requires RNA primers attaching ahead on the lagging strand). The telomeres are disposable buffers at the ends of chromosomes which are truncated during cell division; their presence protects the genes before them on the chromosome from being truncated instead. The telomeres themselves are protected by a complex of shelterin proteins, as well as by the RNA that telomeric DNA encodes (TERRA). Over time, due to each cell division, the telomere ends become shorter. They are replenished by an enzyme, telomerase reverse transcriptase

How is transcription terminated?

There are no specific sequences that stop RNA pol For many genes, termination occurs at various positions in different transcripts Termination is linked to cleavage that is occurring downstream of the polymerase It is linked to the poly A tail and mRNA downstream. The cut for cleavage is made by an enzyme (exonuclease) which is tethered to the RNA Pol II Once the cut has produced a free RNA end, exonuclease moves along the the RNA 5'-3' and a race occurs between this and RNA pol II eventually stopping transcription. :Genes correspond to single transcription units, starting from the promoter and ending at the terminator. Terminating gene transcription is directly coupled to mRNA processing, which occurs cotranscriptionally. When RNA polymerase II (Pol II) reaches the gene end, it first slows down over the terminator. This is partly because 3′-end cleavage and polyadenylation (CPA) complex is recruited onto Pol II when poly(A) signals appear in the nascent transcript. This nascent transcript will often invade the DNA duplex to form an R-loop structure, which induces further polymerase slowdown. During this time, CPA releases mRNA from chromatin into eventual cytoplasmic translation. Pol II continues to transcribe its DNA template after mRNA release. However, this is short-lived, as an exonuclease (Xrn2) degrades the transcript from its 5′ end. When this molecular torpedo catches up with Pol II, then conformational shockwaves are transmitted into its active site, which releases Pol II from the DNA template. Pol II is then free to restart transcription on another gene promoter"

What are co-activators and co-regulators?

These co-regions influence promotors without binding to DNA. Many factors glue together different proteins but do not bind directly to DNA Between enhancers, promotors and co-regulatory elements - they all control the ACTIVITY of a promotor... however they do not control why a given region is transcribed over the other. This is the role of chromatin configuration.

What is ATP dependant chromatin remodelling?

These regulate gene expression by either moving, ejecting or restructuring nucleosomes These protein complexes have a common ATPase domain and energy from hydrolysis of ATP allows these remodelling complexes to reposition This may then create a nucleosome free region of DNA for gene activation Modification of ATP changes the nucleosome occupancy of DNA - by moving nucleosomes along the DNA This movement can affect gene expression by allowing promotor sites to line up in areas with less nucleosomes

What must happen BEFORE the basal transcription apparatus is assembled?

This DEPENDS on a favorable chromatin environment Different transcription machinery will assemble on the DNA at the promotor There are lots of different components which bind to a TATA box.

What is the 5' UTR? Where does it begin and end? Is it translated?

This contains elements for controlling gene expression by way of regulatory elements. The 5′ untranslated region (5′ UTR) (also known as a leader sequence or leader RNA) is the region of an mRNA that is directly upstream from the initiation codon. 5'UTR can be translated or untranslated. If untranslated - it still regulates translation within the gene. It begins at the transcription start site and ends one nucleotide before the start codon of the coding region.

What is a B recognition element?

This is a DNA sequence that is found In most promotor regions in genes in eukaryotes It is often situated near TATA boxes The transcription factor II B (TFIIB) recognizes either BRE and binds to it. Both BREs work in conjunction with the TATA box (and TATA box binding protein), and have various effects on levels of transcription.

What is a topologically associating domain?

This is a self-interacting genomic region, meaning that DNA sequences within a TAD physically interact with eachother more frequently than outside the TAD They are 3D chromosome structures It is long range chromatin interactions So genes from the same or different chromosomes which need to be transcribed at a specific site and time branch out into these TADs. Genes within TAD loops may extend and locate to "transcription factories" within the interchromatin space to be expressed. This may take place intrachromosomally and interchromosomally.

What is the histone code hypothesis?

This is that the transcription of genetic information encoded in DNA is in part regulated by chemical modifications to histone proteins, together with other modifications such as DNA methylation Basically, combinations of histone tail modifications carry information content or act as a recognition platform for effector proteins Different histone modifications lead to active or inactive chromatin

How is RNA pol II brought to the right region at the right time?

This is the function of transcriptional regulatory elements is mediated by proteins known as "transcription factors" In Eukaryotic genes, the regulatory elements exist and function from within a chromatin context • For proteins to be made, transcription factors must have access to genes and their regulatory elements within the chromatin environment of the interphase cell nucleus

What is the point of chromatin remodelling?

Transcription, DNA replication, repair and recombination are dynamically carried out at the chromatin level. Chromatin remodeling represents a change of nucleosome position and conformation, leading to chromatin assembly and disassembly. Chromatin remodeling plays a central role in the regulation of gene expression by providing the transcription machinery with dynamic access to an otherwise tightly packaged genome.

From here, how is RNA pol II activated and start moving? In what direction does it move?

Unlike DNA pol, RNA pol do not need primers to get started. Further action of transcription factors are required before transcription can start TFIIH uses energy from ATP hydrolysis to open up the DNA strand, and allow RNA pol II access to the template strand. RNA polymereases synthesise the RNA strand from nucleotide precursors (ATP, CTP, GTP, UTP) Elongation involves the addition of the appropriate monophosphate residue (AMP, CMP, GMP, UMP) to the free 3' hydroxyl group at the 3' end of the strand. These residies are made from splitting a phosphate group from XTP precursors.

What is FISH? What is the purpose of FISH? What are the key steps in FISH?

his allows you to see DNA or RNA in a cell You can see where the DNA/RNA is within the cell, within the nucleus FISH is a molecular cytogenetic technique that uses fluorescent probes that bind to only areas of the chromosome with a high degree of sequence complementarity *Process of FISH:* Homogenous DNA probes are hybridised to fixed metaphase or prometaphase chromosome spreads on a glass slide Hybridisation of a probe to the DNA is marked as a double spot, indicating the labelled probe has bound to both sister chromatids. This can then be imaged

Describe the properties of heterochromatin Methylation patterns Open or closed? Location in nucleus?

• Condensed/closed chromatin • Transcriptionally inactive • Solenoid/fractal structure and histone H1 rich • Higher order folding • Hypermethylated DNA • Hypoacylated histones • Late DNA replication • Peripheral nuclear location • It's very compact so the cell machinery doesn't have access • Dimethylated and Trimethylated H3K4 gene

Describe the properties of euchromatin Methylation patterns Open or closed? Location in nucleus? High or low concentration of Histone H1?

• Decondensed/open chromatin • Extended beads on string appearance • Histone H1 depleted • Hypomethylated DNA • Hyper-acylated histones • Early replicating DNA Interior nuclear location

What is the histone modification Lysine 4 on histone H3 associated with?

• Lysine 4 on histone H3 it can by mono, tri or bi-methylated. Trimethylation of histone H3 is associated with active transcription, often found at promotors. Other different patterns have different roles.

What are the common post translational modifications occurring to histones?

• You have plenty of different histones • They have a core structure which the DNA wraps around, but you also have this long N-terminal tail that can be post-translationally modified • These contain a number of amino acids that can be modified ○ Lysine residues which can be acetylated and methylated ○ Arganine residues which can be methylated ○ Ubiquinated, phosphylation. • All of these different marks have a different function • This forms a 'histone code'. A single modification that can be sitting near a genome functioning element such as a telomere will all have very specific histone modifications associated with it. Because there are so many different modifications, there are lots of different enzymes involved.