Chapter 7 Control of gene expression

Helix-loop-helix proteins

-have a dimerization domain -extensions of the alpha helices are the recognition helices

CpG islands are associated with housekeeping genes

60% of all protein-coding genes have CpG islands covering their promoters. CpG islands recruit transcription factors and histone modifiers that keep the promoters open and RNA polymerase II at the promoter even if genes are not transcribed

CpGs evolutionarily retained in 'CpG islands'

>200 bp (typically 300-3000 bp); GC% >50 %; observed-to-expected CpG ratio > 60 % CpGs are typically 10 x fold enriched relative to the rest of the genome 20,000 CpG islands in the human genome

2) A researcher studies the expression of the DKK-1 gene in different cell types, and compares DKK-1 expression to the expression of a 'housekeeping gene'? What is a housekeeping gene? Bonus question: Why compare DKK-1 and housekeeping gene expression?

A housekeeping gene is a gene expressed in most cell types and encodes genes necessary for basic functions such a transcription, translation, cytoskeleton and so forth. Housekeeping genes are generally expressed at approximately the same levels across cell type. For this reason one can normalize expression of a target gene, for example DKK-1, to the expression of a housekeeping gene.

5) What is a transcriptional co-regulator and how is it recruited to DNA?

A transcriptional co-regulator is a protein which is recruited to regulatory elements and assists in regulating transcription (remodels chromatin, modifies histone) , but is does not contact the DNA directly. Co-regulators typically bind indirectly to DNA by tethering to other proteins such as transcription factors.

β-sheet DNA requction protein

A two-stranded β-sheet forms the interaction interface with the major groove. A β-sheet consist of β-strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet

Transcription attenuation:

Abort nascent RNA.

7) Cell A expresses isoform 1 of a given gene, whereas cell B expresses isoform 2 of the same gene. Why?

Alternative splejsing - Contributes to protein diversity.

Control of alternative splicing

Alternative splicing is regulated by proteins that bind to the mRNAs in the nucleus and interact with the spliceosomes. Genes are now considered to be the coding and regulatory sequences that direct the production of one or more related proteins or RNAs

leucin zipper

An amphiphatic α-helix in each monomer interacts to form a short coiled-coil structure through hydrophobic interactions between aa (often leucine).

12) What is cell memory and what mechanisms can help to maintain it?

Cell memory is the ability of daughter cells to know what genes the parent cells expressed and thereby maintain the same cell type. It can be maintained by logical loops, such as positive feedback where TF A activates its own expression. This means that when proteins are distributed between daughter cells, the expression of A will be maintained.

1) In your body osteoblasts cells (bone cells) produce bone tissue but cannot store fat, while adipocyte cells (fat cells) store fat but cannot produce bone tissue? Why?

Cell types are differentiated by the amount of RNA, proteins and their modifications. Osteoblast and adipocytes in your body share the same DNA, but express different genes. Approximately 30-60% of genes are expressed in any given cell type, which means that a lot of genes are expressed in widely different cell types. Alternative splicing contributes as well.

Squelching

Competition for co-activators (squelching)

Methylation occurs at

CpG islands and catalysed by DNA methylases.

1) How is eukaryotic DNA methylated? Explain how inheritance in daughter cells is ensured.

DNA can be methylated on cytosine in the sequence CpG. DNA methylation is typically present on both strands due to an enzyme called maintenance methyl transferase, which recognizes a methylated cytosine on one strand and methylates the paired sequence on the other strand. This ensures inheritance, such that after replication one strand in each daughter cell will be methylated in the same pattern as the parent cell. Methylation can also be removed and added de novo, thereby allowing for strong developmental control.

9) In eukaryotic cells, the DNA is ordered in a highly hierarchical chromatin structure within the nucleus, which provides several layers of gene regulation. In contrast, prokaryotes have only one circular 'chromosome' that, besides coiling and supercoiling, possesses no higher structure. How does prokaryotes regulates their gene expression in comparison to eukaryotes?

Eukaryotes: Chromatin is repressive in nature, thus the transcriptional regulation depends on activation by making the DNA accessible. Hisone modification and transcription factors with strong motifs can penetrate heterochromatin and facilitate binding of additional TFs. This facilitates co-factor recruitement and transcriptional activation. Is depenendent on several distal regulatory regions(enhancers) and Mediator complex bridging 3d structure between enhancer and promotor. Prokaryotes: Due to no repressive structure, the transcriptional regulation of prokaryotes is highly dependent on repressive functions. In contrast to eukaryotes, they rely on proximal promotor regions. An example would be the LacZ operon.

10) Glucocorticoid hormone affects gene expression by activation of the glucocorticoid receptor (GR). You want to investigate the effect of glucocorticoid hormone on the expression of 10 potential target genes in cultured mouse hepatocytes and mouse fibroblast cells. Briefly describe how you would set up this experiment Your findings are as follows: In hepatocytes 8 genes are induced and 2 are unchanged, and in fibroblasts 3 genes are induced and 7 genes are unchanged in response to glucocorticoid treatment. Explain why the genes are affected differently by glucocorticoids in the two cell types.

Experimental setup: Cultured cells are treated either with glucocorticoids, or with a vehicle (glucocorticoids are often dissolved in ethanol, therefore this would be a suitable vehicle) for a fixed time period, for example 1 hour. Subsequently, gene expression in glucocorticoid-treated cells is compared with expression vehicle-treated cells, by measuring RNA levels by quantitative PCR. Gene expression is differentially affected in the two cell types because GR acts in concert with other transcription factors to regulate enhancers. Therefore target genes in a particular cell type are the dependent on the combination of transcription factors expressed by that cell. Hepatocytes and fibroblasts express different combinations of transcription factors therefore the response to glucocorticoid treatment is different in the two cell types

What is genomic imprinting and what mechanisms contribute to genomic imprinting?

Genomic imprinting is the process whereby only either the maternal or the paternal allele is expressed and the other is silenced. DNA methylation helps to control this by a variety of mechanisms. E.g. methylation of a promoter can silence the gene or it can silence an insulator such that enhancers can work across insulators.

Specialized cells can be de-differentiated to iPS cells

Induced pluripotent (iPS) cells are similar to embryonic stem (ES) cells

Leaky scanning: .

Initiation can happen at different start codons, as the nucleotides surrounding the start site does not allow proper recognition. This can also be regulated by the different elongation factors which are more or less promiscuous.

Master transcriptional regulators regulate many and overlapping sets of genes.

Master transcriptional regulators regulate the expression each other.

Mediator

Mediator is a large multi subunit complex consisting of 30 subunits It bridges from enhancer to promoter and facilitates assembly of the preinitiation complex

3) How is the methylation status correlated to differentiation capacity? How can a nuclei with highly methylated DNA in nuclei transplantation assays give rise to a new organism?

Methylation status and different capacity are inversely correlated. The more specialized a cell is, the more DNA methylation are present. Methyl transferases methylate DNA, while other proteins are capable of demethylating DNA. Thus, under the right circumstances DNA methylation can be reversed to increase a cells differentiation capacity.

Transcription factors recognize specific sequences in DNA

Most make the majority of their interactions with the major groove. Many weak interactions (~20) → ↑specificity. Interactions consist of hydrogen bonds, electrostatic and hydrophobic interactions. Many transcription factors bind as dimers → ↑specificity

5) What is X chromosome inactivation and what is the mechanism of inactivation?

One of the X chromosomes in fetal embryos (at 100 cell stage) is randomly selected and silenced, and maintained throughout life as silent. A lncRNA called Xist is expressed from a locus on the X chrosome. Xist spreads to the majority (90%) of the X chromosome recruits co-repressors and induces a stable repressive chromatin structure.

Zinc finger protein

One or more zinc atoms as structural component. Zinc finger domains are often found in tandem repeats → ↑specificity

Describe how DNA methylation can repress transcription and the role of reader and writer proteins in this.

One way by which DNA methylation represses transcription is by directly interfering with DNA:protein interactions. Methyl groups on methylated cytosine bases lie in the major groove where transcription factors often contact the DNA. By blocking binding of transcription factor needed for initiation of transcription, methylation can repress gene transcription. Furthermore, methylated CpG recruits Methyl CpG binding proteins (readers) that act as scaffold proteins for co-repressor complexes containing histone modifying enzymes such histone deacetylases and DNA methylases (writers). This leads to spreading of the repressive marks because neighboring nucleosomes and CpGs are modified and this results in repression of gene loci.

9) What is RNA editing, which types of RNA editing exist? How can RNA editing give rise to new isoforms?

RNA editing is an alteration in the nucleotide sequence of a transcript. There are two types - the common A-to-I and the less common C-to-U editing. A-to-I is very prevalent in humans and is controlled by formation of self-complementary stem loops in transcripts. This signifies editing by the ADAR enzymes. Editing can change the amino acid sequence of the resulting product or it can lead to the formation of a new stop codon to allow production of a truncated form of the protein.

Regulation of RNA editing by RNA-binding protein ADAR

RNA editing: Posttranscriptional change of nt sequence. • Deamination 1: A-to-I editing (A•T > I•C). • Deamination II: C-to-U-editing (C•G > U•A). • If exonic: Change of AA sequence (STOP, truncation). • Catalysed by RBP ADAR recognizing dsRNA. • Editing also occurs frequently in microRNAs. • Hyperediting of viral RNA leads to nuclear decay.

The combination of transcriptional regulators determine what genes are active

Requirement for combinations of transcription factors each of which may not be specific for a given cellular state → high degree of specificity

6) What is a riboswitch, and how do riboswitches help the cell to adjust gene expression to levels of specific metabolites? In which cells are riboswitches common?

Riboswitches are short RNA sequences present in the growing mRNA which form secondary structures and upon binding of a small molecule can alter conformation to affect positively or negatively transcription or translation of the mRNA. They thus represent a way for the cell to adapt gene expression to the presence of specific metabolites. Riboswitches are most common in bacteria

7) Describe briefly different mechanisms by which transcriptional repressors can decrease transcription?

Signal is turned off TFs inactivated. TFs or co-repressors can recruit chromatin remodelers or histone modifiers to create a more closed chromatin structure, which does not allow transcription. Another mechanism for gene repression is squelching, in which competition for co-activators between transcription factors results in re-distribution of co-activators from existing enhancers to newly formed enhancers.

Chromatin modifying co-factors have many different functions

Some chromatin modifications are short-lived, and help the cell change transcriptional output. Some (combinations of) modification are long-lasting and help the cell remember which genes have been transcribed.

Transcription factors can act synergistically

Synergy: the combined effect is larger than the sum of the effects

3) How do transcription factors bind to DNA?

TFs recognize the major groove surface using multiple interactions (hydrogen, ionic and hydrophobic) rather than the actual base pairs. However, sequence specificity is achieved as the surface features are sequence-dependent. TFs use different structural motifs, commonly involving either α-helices contacting DNA (HTH, Homeodom ain, bZip, HLH), zinc fingers or β-sheets.

8) In response to stimuli, B lymphocytes switch from expressing a membrane bound antibody to expressing a secreted version of the antibody. Describe how B lymphocytes make this switch in expressing antibody variants. Why are levels of CstF (cleavage-stimulating factor) important for the switch?

The gene encoding the antibody has two RNA cleavage sites/polyadenylation sites which are recognized by the cleavage-stimulating factor (CstF) - an upstream weak binding site and a downstream strong binding site. In unstimulated B lymphocytes where CstF levels are low, only the strong downstream binding site is bound by CstF and a long transcript which encodes the membrane bound variant of the antibody is formed. Stimulation of B lymphocytes leads to upregulation of CstF, thereby increasing RNA cleavage at the upstream weak site. This results in increased levels of the short transcript encoding the secreted version of the antibody. Thus, by controlling CstF levels, the cell can control which RNA cleavage site is used and therefore which antibody variant is expressed.

Gene expression is regulated at many different levels

The most important regulation of gene expression occurs at the transcriptional level.

8) How is developmental control of gene expression achieved?

The specific combination of transcription factors and their activities determine developmental control. For example: In drosophila embryos (single giant cell, multiple nuclei) cis-regulatory elements of the Eve gene is recognized by both transcriptional activators and repressors. The transcriptional outcome is determined by the concentrations of these transcriptional regulators.

uORFs:

These are non-function sequences in the 5' UTR that can trap a ribosome and thereby block translation. Regulation of how efficiently these are recognized allows regulation of translation initiation

Homeodomain proteins

Three α-helices tightly packed by hydrophobic interactions . Helix 2 and 3 are very similar to helix-turn-helix proteins connected. The recognition helix makes contacts with in the major groove.

Dimerization increases affinity and specificity

Transcription factors are promiscuous in their binding - bind to many variants of a motif. Transcription factor affinity and specificity is increased by dimerization.

6) What sort of mechanisms can activators use to increase transcription?

Transcriptional activators ... ... promote binding of additional transcriptional regulators. ... recruit and position the RNA polymerase ... release a positioned polymerase, initiating transcription. ...release stalled polymerases.

Helix-turn-helix proteins

Two α-helices connected with a short chain of aa (turn). The recognition helix makes contacts with the major groove

Writers

catalyze formation of covalent modifications

Spontaneous C→U deaminations are

corrected

Histone acetylation generally

decreases interaction between nucleosome and DNA. Which makes DNA more accesible for transkribtion.

Cell type selective/specific genes:

expressed at very different levels in different cell types

Housekeeping genes:

genes are expressed in all/most cell types

What is an iPS cell?

iPS cells stand for Induced Pluripotent Cells, and are non-stem cells which have been dedifferentiated by expression of master transcriptional regulators of pluripotency (the so-called Yamanaka factors - Oct4, Sox2, Klf4 and c-Myc).

Cells utilize DNAme to establish

inactive chromatin.

Transcription factors bind to enhancers far away from promoters and

interact with the promoters through co-factor complexes and DNA looping.

10) What is mRNA localization, and why is it useful for the cell? What determines where RNA´s are transported?

mRNA localization is the process where an mRNA is transported to a specific compartment of the cell prior to being translated, rather than being translated immediately after nuclear export. The advantages are e.g. that this allows proteins to be produced where they are needed, that it allows for cytosolic asymmetry to be generated, and it allows for protein expression to be regulated independently in different regions of the cytosol. Signals determining the localization of a RNA is often present in the 3'UTR (from stop codon to start of poly(A) tail).

Spontaneous 5me-C→T deaminations are

not corrected

Posttranscriptional gene regulation

occurs at all the steps from RNA processing to protein modification

Transcription factors must

overcome inhibitory effects of nucleosomes.

Readers

recognize modifications

Erasers

remove covalent modifications

DNAme marks

repressed chromatin.

Reader cooperates with histone modifier that introduces the same mark as the reader recognizes. This leads to

spreading of the mark.

iRNA-seq can be used to determine

transcriptional changes based on total RNA-seq

mRNA localisation in subcellular domains

• After nuclear pore exclusion, mRNA is met by ribosomes. • Ribosome localisation governs ER vs secretion by SP 'ZIP'. • mRNAs also be specified to intracellular locations. • mRNA 'ZIP' enables local translation and protein gradients. • RNA sequences often reside in the mRNA 3'-UTR. • Fruitfly: Bicoid TF mRNA gradient defines anterior embryo. • mRNA localisation involves on cytoskeletal (MT) transport.

Riboswitches are functional RNA modules.

• Moduls often situated at 5'-end of transcripts. • Bypass need for regulatory proteins (economical). • Binding affinity matches small molecule•protein. • Pervasively found in prokaryotes. • HIV TAT overrides TA by recruiting a RBP.

Possible (cellular) fate of mRNA molecules

• SG and PB: Functional 'organelles' without membrane. • Decapping and RNA degradation: P-bodies. • Salvage of mRNAs from PB by inclusion in SG. • SG make mRNAs translation-competent again. • Competence via exchange of ribonucleoproteins. • Cellular 'starving' and non-translated mRNA saved. • Rationale: Cell 'invested' in proper mRNA maturation.

Competition between mRNA translation and decay

• The mRNA 3'-UTR sequence dictates lifetime. • RBPs on 3'UTR control increase or decrease the rate of pA shortening. • Factors driving translation efficiency inherently decelerate mRNA decay

Influence of mRNA stability on gene control

• mRNA halflife in eukaryotes from 30m to 10h. • Proteins with rapid changes (GF, TF) short halflife. • Degradation start simultanously at 3'-end (pA tail). • Once pA tail deceeds critical length (25nt in human): • Decapping (5') • Continued pA degradation (3') • Specific case: Endoribonuclease activity > rapid deday.

Requirement for combinations of transcription factors each of which may not be specific for a given cellular state

→ high degree of specificity

Total RNA-seq

→ summarize intron reads for each gene → normalize to read length → amount of native transcript ≈ ongoing transcription