p53

Learning Goals/Objectives for Chapter 5D:  After class and this reading, students will be able to describe general mechanisms of how a gene for a given protein might be negatively and positively regulated at the level of gene transcription; describe the structure/function/role of promoters, response elements, RNA polymerase, transcription factors, nucleosomes, histone proteins, epigenetic modifications of DNA in gene transcription; explain the differences (structural, Kds) between specific and nonspecific binding of a ligand to a macromolecule, at the structural level; describe the structural features of both proteins and DNA that result in specific and nonspecific binding; describe and give examples of how post-translational modifications of proteins and epigentic modifications of DNA can alter gene expression; explain how the apparent Kd for a protein binding to DNA can be altered by the presence of another protein bound to DNA at a proximal site describe the basis of RNA interference in gene expression

Imagine you have been given a string approximately 3 feet long, which represents an unwound, deproteinated, human chromosome. In actuality, such naked DNA does not exist in the cell nucleus.  Rather, it is wound around a series of positively charged histone proteins; in the electron microscope it resembles beads on a string. This structure is wound into a cylindrical "solenoid" structure which is further packaged to fit into the nucleus along with the rest of the chromosomes. There just happens to be a small dot in the dead center of the string you have been given. It represents the gene for a particular protein called metallothionein. This gene is expressed and the protein metallothionein is made when cells are exposed to heavy metals like Cd. Your job today is to figure out a possible way (there are many) in which the cell would not express the gene, or express it to a small, constituitive level, in the absence of Cd exposure, and how the gene might be activated - through transcription of the gene and translation of  the resulting mRNA to form the metallothionein protein upon exposure to Cd. (Hint: Cd doe not DIRECTLY bind to DNA.) Propose mechanisms to address the following questions. Do not propose any magical mechanisms:

1. How might gene expression be repressed in the absence of Cd. List some possibilities.
2. How might gene expression be activated in the presence of Cd

One of the central questions of modern biology is what controls gene expression. As we have previously described, genes must be "turned on" at the right time, in the right cell. To a first approximation, all the cells in an organism contains the same DNA (with the exception of germ cells and immune cells). Cell type is determined by what genes are expressed at a given time. Likewise, cell can change (differentiate) into different types of cells by altering the expression of genes. The central dogma of biology describes how genes are first transcribed to messenger RNA (mRNA), and then the mRNA is translated into a corresponding protein sequence.   The links below should be reviewed by those who have little background on the Central Dogma of Biology and of the nature of a gene.

• overview of Central Dogma of Biology for the non-biochemistry major
• A view of genes and their products:  Simplicity to Complexity

Jmol: Simple DNA Tutorial 

Proteins can then be post-translationally modified, localized to certain sites within the cells, and ultimately  degraded. If functional proteins are considered the end-product of gene expression, the control of gene expression could theoretically occur at any of these steps in the process.

 Figure:  PROCESSES THAT AFFECT THE STEADY STATE CONCENTRATION OF A PROTEIN 

 

Mostly, however, gene expression is controlled at the level of transcription. This makes great biological sense, since it would be less energetically wasteful to induce or inhibit the ultimate expression of a functional protein at a step early in the process. How can gene expression be regulated at the transcriptional level? Many examples have been documented. The main control is typically exerted at the level of RNA polymerase binding just upstream (5') of a site for transcriptional initiation. Other factors, called transcription factors (which are usually proteins), bind to the same region and promote the binding of RNA polymerase at its binding site, called the promoter. Proteins can also bind to sites on DNA (operator in prokaryotes) and inhibit the assembly of the transcription complex and hence transcription. Regulation of gene transcription then becomes a matter of binding the appropriate transcription factors and RNA polymerase to the appropriate region at the start site for gene transcription. Regulation of gene expression by proteins can be either positive or negative.  Regulation in prokaryotes is usually negative while it is positive in eukaryotes.

Figure:  Positive and Negative Regulation of Gene Transcription

 

CONTROL OF GENE TRANSCRIPTION IN PROKARYOTES: THE E. COLI LAC OPERON

The regulation of the genes involved in lactose utilization won Jacob and Monod (of MWC fame) the Nobel Prize. Lactose can be used as the sole source of carbon by E. Coli. Three genes are required for lactose utilization, beta-galactosidase (lac Z, cleaves lactose to Gal and Glc), galactoside permease (lac Y, transports Lac into the cell) and thiogalactoside transacetylase (lac A, function unknown). These genes follow one another on the DNA, and have 1 promoter region. On transcription and translation, one long poly-protein is made, which is cleaved post-translationally to form the individual proteins.

Figure:  FUNCTION OF PROTEINS IN GALACTOSE UTILIZATION  

 

In addition, another gene, the Gal repressor, is found just upstream of the Gal utilization genes. It has its own promoter (P_I).  A gene cluster, including promoter and any regulatory DNA sequences is called an operon, for example, the Lac operon. In this case, transcription from the operon is induced in response to a molecular signal - i.e. the presence of lactose, or allolactose. The signal binds to the repressor protein, which is bound to the operator DNA, which in the absence of the signal inhibits transcription. When the signal, in this case allolactose or another beta-galactosides, such as isopropylthiogalactoside (IPTG), binds to the repressor protein, a conformation change occurs in the repressor, resulting in a higher Kd for the operator DNA, and subsequent dissociation of the repressor-galactoside complex. Transcription ensues.

Figure:  IPTG and Lactose Structures

 

IPTG is an inducer of the lac operon but is not a substrate for the enzymes produced. We will use IPTG to induce expression of human adipocyte acid phosphatase b (HAAP-b) in lab 4B. The plasmid containing the gene for HAAP-b has been engineered to contain the lac promoter just before the start site for gene transcription. By adding IPTG to the growing cells, the cells can be induced to synthesize the protein, HAAP-b.

Figure:  INDUCTION OF LAC OPERON

 

Many analogous but distinct methods are used to control gene transcription in prokaryotes. The control of lac operon transcription is but one example.

CONTROL OF GENE TRANSCRIPTION IN EUKARYOTES

Three major differences exists in the control mechanisms used to regulate gene transcription in eukaryotes compared to prokaryotes.

• multiple changes occur in the structure of chromatin at the site of transcription
• positive mechanisms regulate transcriptions much more often than negative ones.
• transcription and translation occur at spatially and temporally distinct sites and times.

The genomes of eukaryotes are much larger than prokaryotes. This poses some problems with respect to binding. Remember, DNA binding proteins demonstrate both nonspecific and specific binding. Nonspecific binding may help a protein find a specific site in the genome, but as the size of the genome increases, the chance of finding multiple specific sites randomly distributed increases. This problem can be avoided if multiple proteins are required to generate an active transcription complex. The chance of finding two or more specific sites for different proteins in proximity at sites other than required for gene transcription are very low. Multiple negative regulators would not be needed since just the binding of one regulator would probably be sufficient. Most eukaryotic genes have about 5 regulatory sites for binding transcription factors and RNA polymerase. Examples of these transcription factors are show in the figure below.

Figure:  Control of globin gene transcription

 

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Figure:  Example of transcription complexes

 

Light can even regulate gene expression by indirectly activating an inactive transcription factor in plants.  The  transcription factor PIF3 binds to a promoter region (G-box) of light-responsive genes. Only when PIF3 binds another transcription facotr, Pr, is transcription activated.  Pr, a "photoreceptor" is found in an inactive form in the cytoplasm.  When it absorbs red light, it undegoes a conformational shift and moves into the nucleus, where it can bind PIF3 and activate transcription.  The activation complex is inactivated when Pr interacts with far-red light.

Figure:  Upon absorbing red light, a phytochrome photoreceptor is converted from the inactive Pr form to the active Pfr form

 

• p53 information from the HHMI
• animation: p53 and gene transcription from the HHMI (loads slowly)

STRUCTURAL FEATURES OF SPECIFIC DNA BINDING SITES

Since RNA polymerase must interact at the promoter site of all genes, you might expect that all genes would have a similar nucleotide sequence in the promoter region. This is found to be true for both prokaryotic and eukaryotic genes. You would expect, however, that all transcription factors would not have identical DNA binding sequences.   The sequences of DNA just upstream of the start site of the gene that binds protein (RNA polymerase, transcription factors, etc) are called promoters.  The table below shows the common DNA sequence motif called the Pribnow or TATA box found at around -10 base pairs upstream from the start site, and another at -35.  Proteins bind to these sites and facilitate binding of  RNA polymerase, leading to gene transcripton.

Prokaryotic  Promoter Sequences

Promoter	-35 Region	Spacer	-10 Region	Spacer	RNA start
trp operon	TTGACA	N17	TTAACT	N7	A
tRNAtyr	TTACA	N16	TATGAT	N7	A
lP2	TTGACA	N17	GATACT	N6	G
lac operon	TTTACA	N17	TATGTT	N6	A
rec A	TTGATA	N16	TATAAT	N7	A
lex A	TTCCAA	N17	TATACT	N6	A
T7A3	TTGACA	N17	TACGAT	N7	A
consensus	TTGACA		TATAAT

The TATA box in prokayotes is also known as the Pribnow box.  A similar sequence is found in eukaryotes (consensus TATAAA) located about 25 nucleotides upstream from the transcription start site.  It is also called the Goldstein-Hogness box.

Jmol: TATA Box Binding Protein 

In addition, in eukaryotes, sequences further upstream called response elements bind specific proteins (such as CREB or cyclic AMP response element binding protein) to further control gene transcription.

Eukaryotic Response Elements  (RE)s

Regulatory agent	Module	Consensus	DNA bound	Factor	Size (daltons)
Heat Shock	HSE	CNNGAANNTCCNNG	27 bp	HSTF	93,000
Glucocorticoid	GRE	TGGTACAAATGTTCT	20 bp	Receptor	94,000
Cadmium	MRE	CGNCCCGGNCNC	.	?	.
Phorbol Ester	TRE	TGACTCA	22 bp	AP1	39,000
Serum	SRE	CCATATTAGG	20 bp	SRF	52,000
Antioxidant	ARE	GTGACTCAGC
Pheromone (fungus)		ACAAAGGGA
Hypoxia	HRE	CCACAGTGCATACGT GGGCTCCAACAGGTC CTCTCCCTCCCATGCA		Hypoxia Inducible Factor	826 aa
Peroxisome Proliferator Activated Receptor (PPAR)	PPRE	aGG_CAAAGGT(CG)A		PPAR	59,000
Steroid (general) (progesterone, androgen, mineralcorticoids, glucocorticoids		AGAACAxxxACAAGA (inverted repeat)

Proteins can interact specifically with DNA through electrostatic, H-bond, and hydrophobic interactions.  AT and GC base pairs have available H bond donors and acceptors which are exposed in the major and minor grove of the ds DNA helix, allowing specific protein-DNA interactions.

Figure:  AT and GC base pairs have available H bond donors and acceptors

 

Jmol: Simple DNA Tutorial (see last selection buttons to see H bond donors and acceptors in the major grove.

Jmol: RNA Polymerase II/DNA/RNA complex.

 

Gene Transcription, Proteolysis and Membrane Lipids

An interesting example of transcriptional control occurs to maintain the balance of lipids in biological membranes.  The phospholipids and sphingolipids in membranes are extremely heterogeneous, owing to the diversity of head groups and acyl chain composition.  Given this great diversity, it is remarkable the different cells are able to maintain the specificity of lipid types in different cells, in different membranes within cells, and within a given leaflet of a membrane (remember our discussion of lipid rafts).  How can the cell regulate the type of lipids that it synthesizes?  What controls the transcription of genes for lipid synthesis?

Regulation of transcription of these genes appears to be controlled by multidomain proteins that bind to sterol response elements in the DNA.  The proteins, called Sterol Response Element Binding Proteins (SREBPs) are activated by proteolysis to release a transcription factor domain which migrates to the nucleus.  Proteolysis of SREBP occurs in the Golgi by resident proteases.    The SREBP in the Golgi is in complex with another protein, SREP cleavage-activating protein (SCAP), which facilitates movement of the  SREBP to the Golgi from its site of synthesis in the endoplasmic reticulum.  Lipid regulation occurs when fatty acids, cholesterol, or PL derivatives like phosphoethanolamine (from ceramide) inhibits proteolytic activation of the SREBP.  Regulation depends on whether or not SCAP "ferries" SREBP to the Golgi.  It appears that SCAP binds to SREP and transfers it to the Golgi membrane, but only when sterol levels are low.   When cholesterol is high, it binds to the transmembrane domain of SCAP and prevents SCAP from interacting with SREP and transferring it to the Golgi.

Auxin, a major plant hormone that induces gene expression, also seems to activate transcription through proteolysis.  When bound to its soluble cytoplasmic receptors, ubiquitin protein ligase SCF^TIR1, it activate proteolysis of protein that inhibit transcription.  

Jmol : Auxin Receptor

STRUCTURAL FEATURES OF DNA-BINDING PROTEINS

Not any protein can bind specifically to DNA. Analysis of DNA binding proteins shows common motifs are found among them.

• helix-turn-helix: found in prokaryotic DNA binding proteins.

Figure:  helix-turn-helix

 

The figures shows two such proteins, the cro repressor from bacteriophage 434 and the lambda repressor from the bacteriophage lambda. (Bacteriophages are viruses that infect bacteia.)  Notice how specificity is achieved, in part, by the formation of specific H-bonds between the protein and the major grove of the operator DNA.

Figure:  Lambda Repressor/DNA Complex

 

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Figure:  H Bond interactions between l repressor and DNA

 

Chime: Lambda-Repressor Complex   Jmol: Lambda-Repressor Complex

• zinc finger: (eukaryotes) These proteins have a common sequence motif of
  X₃-Cys-X_2-4-Cys-X₁₂-His-X_3-4-His-X_4- in which X is any amino acid. Zn²⁺ is tetrahedrally coordinated with the Cys and His side chains, which are on one of two antiparallel beta strands, and an alpha helix, respectively. The zinc finger, stabilized by the zinc, binds to the major groove of DNA. ]

Figure:  zinc finger

 

Chime: Zif268:DNA Complex   Jmol: Zif268:DNA Complex

Zn finger proteins, of which 900 are encoded in the human genome, can be mobilized to actual repair specific mutations in cells, which if carried out in a high enough percentage of mutant cells could cure specific genetic diseases such as some forms of severe combined immunodeficiency disease.  In this new technique (Urnov et al, 2005), multiple linked Zn finger binding domains,  (one of the natural-occurring ones or mutant forms produced in the lab), each one specific for a certain nucleotide sequence, is linked to a nonspeciifc endonuclease, derived from the enzyme FokI.  The nuclease is active in dimeric form so the active complex requires two endonuclease domains, each bound to four different Zn finger domains, to assemble at the target site.  Specificity of binding is achieved by selection by the Zn finger domains.  A nick is then made by the DNA by the nuclease, and host cell repair mechanisms ensue.  This process involves strand separation, homologous recombination of the nicked region with complementary DNA within the cell, and repair of the nick.  If excess wild type (non-mutated) DNA is added to the cells and uses as the template, the normal DNA repair mutation would fix the mutation.  Urnov et al have shown the up to 20% of cultured cells containing a mutation can be repair in the lab.  If these cells gain a selective growth advantage, the mutated cells would eventually be replaced with wild type cells.

• steroid hormone receptors: (eukaryotes) In contrast to most hormones, which bind to cell surface receptors, steroid hormones (derivatives of cholesterol) pass through the cell membrane and bind to cytoplasmic receptors through a hormone binding domain. This changes the shape of the receptor which then binds to a specific site on the DNA (hormone response element) though a DNA binding domain. In a structure analogous to the zinc finger, Zn ²⁺ is tetrahedrally coordinated to 4 Cys, in a globular-like structure which binds as a dimer to two identical, but reversed sequences of DNA (palindrome) within the major grove. (An example of a palindrome: Able was I ere I saw Elba.)
• leucine zippers: (eukaryotes) These proteins contain stretches of 35 amino acids in which Leu is found repeatedly at intervals of 7 amino acids. These regions of the protein form amphiphilic helices, with Leu on one face. Two of these proteins can form a dimer, stabilized by the binding of these amphiphilic helices to one another, forming a coiled-coil, much as in the muscle protein myosin.  The leucine zipper represents the protein binding domain of the protein. The DNA binding domain is found in the first 30 N-terminal amino acids, which are basic and form an alpha helix when the protein binds to DNA. The leucine zipper then functions to bring two DNA binding proteins together, allowing the N-terminal bases helices to interact with the major grove of DNA in a base-specific fashion.

Figure:  leucine zippers (made with VMD)

 

Chime: Leucine Zipper  Jmol: Leucine Zipper

BINDING AND THE YEAST TWO-HYBRID SYSTEM IN STUDYING THE INTERACTOME

Transcription factors must do more than bind to upstream targets (response elements) on DNA.  They must also interact with and activate the transcription machinery, including RNA polymerase and other assembled proteins, for transcription to occur.  To accomplish both tasks, protein transcription factors usually consist of two distinct and often separable domains, a DNA Binding Domain (DNA-BD) and an Activation Domain (AD).  The BD usually contained the DNA binding motifs discussed above.  In a clever feat of genetic manipulation, scientist have taken the part of the transcription factor gene encoding the DNA-BD and fused it to a gene for a protein called the bait protein.  Likewise the other part of the transcription factor gene encoding the AD is fused it to a gene for another protein, the target,  that could bind the bait protein.   Plasmids with the gene constructs are added to yeast.  By themselves, the separated BD and AD can not activate transcription from a gene in the yeast that is inducible by the whole transcription factor.  However, if  the BD-Bait gene and the AD-Target genes are both added on separate plasmids, and both fusion genes are ultimately transcribed and the fusion RNA translated into fusions proteins, then transcription from the inducible gene can occur if the bait and target part domains of the fusion proteins bind to each other, allowing binding of the BD domain and the AD domain to their target sites, leading to gene transcription.   This yeast two-hybrid method has allowed the determination of protein binding partners, part of what is now termed the interactome. 

Figure:  Yeast Two-Hybrid

 

PROBING THE INTERACTOME:  OTHER METHODS Other similar methods have been developed to map bate-trap  protein-protein interactions in yeast.  Protein Fragment Complementation (PFC)  (Tarassov, K. et al):  A gene encoding one fragment of a reporter enzyme, dihydrofolate reductase (DHFR), is fused to a gene for a bait protein and inserted into a plasmid.  A second gene representing the second fragment of a reporter enzyme is linked to possible target protein genes.  In a cell transformed with plasmids, the reporter gene will ultimately display enzymatic activity only if the translated bait protein binds to a translated target protein, allowing the two fragments of the enzyme to interact and fold collectively into a holo-, active enzyme activity.  The reporter gene was a mutant of DHFR that was resistant to an inhibitor, methotrexate.  Functional DHFR activity leads to cell growth in the presence of methotrexate.  Figure:  Protein Fragment Complementation   Tandem Affinity Purification (TAP)  (Rigaut, G. et al) A generic protein purification method for protein complex characterization and proteome exploration.  Nature Biotechnology 17. 1030 (1999):  A gene for a bait protein is linked sequentially to genes encoding two separate tags, one for Protein A (which binds immunoglobulin G - IgG) and a calmodulin binding peptide (which binds the protein calmodulin in a process which requires calcium.  In between the genes for the two tags is a nucleotide sequence encoding a short, protease sensitive linker peptide.  The gene construct is introduced into yeast and expression induced.  Cell are lysed and the extract applied to affinity chromatography beads containing covalently attached IgG, which binds the Protein A tag.  After extensive washing the bound  bait protein, with associated target proteins, is eluted by proteolysis of the peptide linker.  The eluate is applied to a second affinity column containing covalently attached calmodulin.  After washing, the trap and associated target proteins are eluted with a calcium chelator (EGTA or EDTA) as the interaction of calmodulin with the second tag, calmodulin binding peptide, requires calcium.  Eluted target proteins can be identified by 2D PAGE or mass spectrometry.   Figure:  Tandem Affinity Purification     A recent comparison of the Y2H, PFC, and TAP methods was made (Jensen, L. and Bork, 2008;  Yu, H et al, 2008). Not unexpectedly, the Y2H was best at determining nuclear protein interactions, the TAP for cytoplasmic and abundant proteins, and PFC for transmembrane proteins.  All suffer somewhat in identifying transient protein complexes.

PHOSPHORYLATION AND CONTROL OF GENE EXPRESSION

A common way to control gene expression is by controlling the post-translational phosphorylation of transcription factors by ATP.  This modification might activate or inhibit the transcription factor in turning on gene expression.  The added phosphate groups might be necessary for direct binding interactions leading to gene transcription or they might lead to a conformational change in the transcription factor, which could activate or inhibit gene transcription.  A recent example of this later case is the control of the activity of the transcription factor p53.  p53 has many activities in the cell, a primary one as a suppressor of tumor cell growth.  If a cell is subjected to stress that results in genetic damage (an event which could lead a cell to transform into a tumor cell), this protein becomes an active transcription factor, leading to the expression of many genes, including those involved in programmed cell death and cell cycle regulation.  Both of these effects could clearly inhibit cell proliferation.  Hence p53 is a tumor suppressor gene.  p53 is usually bound to the protein HDM2 which down regulates its activity by leading to its degradation.  Stress signals lead to the activation of protein kinases in the cell (such as p38, JNK, and cdc2), causing phosphorylation of Ser 33 and 315 and Thr 81 in p53. This leads to the binding of Pin 1, a peptidyl-prolyl isomerase, which catalyzes the trans<=>cis conformational changes of X-Pro bounds.  Pin 1 appears to bind only when p53 is phosphorylated.  The ensuing change in p53 conformation presumably leads to its activation as a transcription factor.

Figure:  Activation of p53 as a transcription factor by phosphorylation and conformational change

 

POSITIVE TRANSCRIPTION FACTORS - A NEW CLASIFICATION

As inferred from above, transcription factors can be classified based on their protein structure.  A newer classification scheme, based on the function/activity of the transcription factors, has been proposed by Brivan, Lou and Darnell (Science, 295, pg 813, 2002), as illustrated in the flowchart below, along with specific examples. The classes of transcription factors include those that are:

• constitutively active :  are always active in the nucleus of the cell and probably activate transcription of genes that must always be turned on;

The rest must be activated by some means, which include those that are:

• developmental or cell type-specific whose genes must be transcribed (probably in a regulated fashion) to form the transcription factor which then enters the nucleus; 
• signal dependent transcription factors, which are activated through a signaling event.  

There are classes of signal-dependent transcription factors that are activated by:

• steroids, which are cholesterol derivatives that can pass through the cell membrane and bind steroid-specific transcription factors which turn on specific sets of genes; most of these transcription factors are present in the nucleus and are activated there by steroid hormones.  One exception is the glucocorticoid receptor (GR) which is found in the cytoplasm;
• internal signals derived from the cell, such as internally made lipid signals.  
• cell surface receptor-ligand interactions;  

There are two types of receptor-ligand interactions that lead to transcription factor initiation.  

• small ligand molecules (like epinephrine) bind transmembrane receptors which leads to formation of second messengers or signals inside the cell, which ultimately activate Ser-phosphorylation activity.  Nuclear transcription factors can become phosphorylated and activated.  
• small ligands bind transmembrane receptors which then bind to and activate latent transcription factors in the cytoplasm, which then migrate to the nucleus.

Figure:  Transcription Factors:  Functional Classification

 

COOPERATIVE BINDING OF PROTEINS TO DNA

We have just spend much time studying the cooperative binding of oxygen to hemoglobin.  Cooperativity seemed to be require conformational changes in a multimeric protein.   Is it possible to get cooperative binding of ligands without conformational changes?  In a recent book by Ptashne and Gann (Genes and Signals, Cold Spring Harbor Press, 2002), it is argued that you can and through a very simple mechanism.  

It must be clear that to activate gene transcription, several transcription factor proteins must assembly at the promoter before RNA polymerase can transcribe a gene.  There are multiple DNA-protein and protein-protein contacts.  To simplify this discussion, consider the case of two proteins, A and B, that must bind to the DNA and to each other for transcription to occur. 

Figure:  two proteins, A and B

 

The binding of each protein alone is characterized by a characteristic Kd, k_on, and k_off.   What happens to k_on and k_off for protein B, for example, when A is already bound?  You can imagine that k_on doesn't change much, but what about k_off after the protein is interacting both with its DNA site and with protein A?  If B did dissociate from its DNA site, it would still be held in close approximation to that site because of its interaction with the bound protein A.  Its effective concentration goes up and you should readily image that it would rebind very quickly to its DNA site.  The net effect would be that it's apparent k_off would decrease, which would increase its apparent binding affinity and decrease its apparent Kd (remember that Kd = k_off/k_on).  Hence prior binding of A would lead to cooperative binding of protein B.

• Animation showing cooperative binding of B to the protein A-DNA complex.  
• Nuclear receptor Database

EPIGENTIC CONTROL OF DNA TRANSCRIPTION - METHYLATION OF DNA

The fertilized egg is a totipotent cell.  That is, through a series of divisions, its progeny cells can eventually become any of about 200 histologically different cell types. With subsequent cell division in the developing embryo, cells find themselves in different topological environments and have different cell-cell contact.  Through signal transduction through the cell membrane, these cells start to become different, or differentiate, into other cells types.  They do so by activating and inhibiting the expression of a different set of genes to form a different set of proteins in the cells.  Most cells become terminally differentiated and eventually (after maybe a hundred cell divisions) lose the ability to divide and hence begin to die.  However, a few types of cells, called stem cells, retain the ability to differentiate into other cells types in a regenerative process.  These cells are pluripotent in that they can differentiate into other cell types.  

How do dividing cells know what types of genes to actively transcribe?  How can they have "memory" of the cells type they were before division?  This appears to happen without alteration of the nucleotide sequence of the DNA in these cells.  The main mechanism appears to be a inheritable but modifiable pattern of chemical modifications to the DNA (not unlike co- or post-translational modification of proteins) involving methylation/demethylation (by a methylase and demethylase) of  cytosine in CpG dinucleotide repeats in the DNA.  Also proteins can bind to DNA and methylated DNA to modify the course of gene expression in daughter cells.  Such chemical modifications to the DNA which modify gene transcription are examples of epigenetic mechanisms controlling gene expression.  

You are all familiar with the cloning of animals from the DNA of adult cells (from Jurassic Park to the actual cloning of the sheep Dolly).  In this process, the nucleus from an adult somatic cell (like a check epithelial cell) is removed and placed in an egg from which the nucleus has been removed (enucleated).  The egg now has a full complement of DNA, just like an adult cell, but it didn't get the full set by normal means - i.e. by receiving half from a sperm to complement its own normal half.  There is another potentially big problem.  The DNA in the egg has the methylation pattern of a terminally differentiated adult cell.  It must be reprogrammed by undergoing extensive demethylation and remethylation to the "correct" epigenetic methylation state if the egg has a chance to form a normal embryo, fetus, and adult.  Obviously this can happen, as evidenced by Dolly and success in cloning cows, cats, pigs, mice, and dogs.  However, it is very difficult to achieve and probably accounts for the low success rate of cloning.   

Methylation patterns can account for gene silencing (in which one gene in a pair of identical chromosomes is not expressed)  and inactivation of one entire X chromosome in a female (who has 2 X chromosomes).  In general, transcription from genes that are methylated is inhibited.

Jmol: Methyl-CpG-Binding Domain of Human MBD1 in Complex with Methylated DNA 

4/29/09

Methylation patterns are inheritable and are also determined by the environmental variables.  A recent study of neuron specific glucocorticoid receptor gene (NRGC1) promoter in the hippocampus showed an increase in methylation of the promoter and decreased levels of the mRNA transcript from the gene in suicide victims who had a history of childhood abuse compared to suicide victims that didn't suffer such abuse.   These result paralleled those found in rats who were raised in a non-nurturing environment.  The hippocampus is involved in the stress response.

Methylation patterns can be passed onto to daughter cells and on to offspring.  This inheritance depends on specific binding of the DNA methylase to the methylated CpG on the template strand of the newly replicated DNA, which positions it to methylate the complementary newly replicated strand on the C hydrogen bonded to the template strand G.  This enzyme specificity explains the requirement for a CpG dinucleotide methylation site in DNA, as shown below. 

Figure:  Methylation of CpG

 

The dinucleotide sequence CpG is underrepresented in the human genome, yet is found in about 60% of promoter regions for genes.  CpGs in promoter regions that are constitutively transcribed are not methylated.  Methylated DNA outside of genes for proteins help to silence transcription from those regions.  The CpG dinucleotide is probably found in low abundance since if 5 methyl cytosine spontaneously deaminates, it forms thymine which would lead to a CG to AT base pair mutation.

Yet another covalent epigentic modification has been discovered in neurons, where high levels of 5-hydroxymethyl cytosine are found.  A 5-methylcytosine hydroxylase has also been discovered.  Both probably have large roles in gene regulation.

CHROMATIN REMODELING AND GENE EXPRESSION

Control of DNA transcription in eukaryotes was thought to involve the assembly of many proteins at the promoter into a pre-initiation complex (PIC).  Once assembled, RNA polymerase could bind and transcription would be initiated.  But wait a minute!  Isn't DNA packaged in the nucleus into chromatin in which 147 BP of DNA is wound around a core of 4 pairs of positively charged histone proteins - including H2A, 2B, 3, and 4 - to form a nucleosome, seen under a microscope as beads on a string? 

Chime:  nucleosome   Jmol:  nucleosome

Isn't this chromatin further wound into fibers which result in the classic picture of sister chromatids ready to separate at cell division?  How could the transcription factors and RNA polymerase recognize target sites on DNA given this degree of "folding" and condensation of the DNA?

Clearly the complex compacted state of DNA and its interaction with the histone proteins must be "remodeled" to allow interactions of the transcription factors and RNA polymerase (which is about the same size as a nucleosome).  The regulation of this chromatin remodeling clearly affects gene transcription, and is another example of epigenetic changes that can affects phenotype.  The state of chromatin structure is regulated by enzymes that affect histone structure and function by chemically modifying the histone proteins (through acetylation, methylation, and phosphorylation) .  Likewise, the DNA at the promoter region is changed by enzymes that remodel the DNA through an ATP dependent series of modifications.  For example when histones are modified by histone acetyltransferase (HAT's), other modeling factors (SWI/SNF) are recruited to the chromatin.  Chromatin remodeling would also be affected by that cell cycle stage of the cell.  For example, chromatin condensed in sister chromatids ready for cells division would have different remodeling requirements for gene transcription than might chromatin in the form of bead on a string.  Likewise remodeling efforts would also be gene-specific.  

The figure below shows how remodeling is coupled to formation of the pre-initiation complex for three genes:  

• yeast HO gene:  Swi5p activator binding results in the interaction of the SWI/SNF ATP-dependent remodeling enzyme, which leads to the binding of histone acetyltransferase (HAT).  These facilitate formation of the pre-initiation complex.
• human interferon-b gene: gene sequences known as activators, 5' to the promoter, bind HATs.  When histones are acetylated, SWI/SNF interacts to remodel the chromatin and facilitate PIC formation.
• human a-1 antitrypsin gene:  the PIC is preformed and recruits HAT and SWI/SNF, which leads to gene transcription.

Alternations in chromatin remodeling could lead to changes in gene expression, in some cases causing cancer.  SNF5 is a component of the SWI/SNF complex and in its normal form acts to suppress tumors (i.e. its gene is a tumor suppressor gene).  Mutations in SNF5 are associated with rare and aggressive childhood tumors.  Stuart Orkin has developed a technique to alter the gene in some mouse cells to produce an inverted gene which produces no functional SNF5.   Cells with this mutation become tumor cells almost immediately.

Figure:  Remodeling of Chromatin and Control of DNA Transcription

 

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• SNF5 and cancer

DNA winds around the histone core to form the nucleosome. 

However, histone tails not associated with DNA binding protrude from the nucleosome, and the function of these tails is just being unraveled.  The amino acids in these tails are clearly sites for posttranslational modifications, including methylation, acetylation, and phosphorylation.  When modified, these tails would provide additional binding sites for protein which could regulate transcription and  chromatin modeling, thus modifying the "genetic code".  Understanding the "histone code" and how it affects gene transcription becomes important.  For example, the methylation of Lys 9 on histone 3 leads to binding of heterochromatin-associated protein, leading to inhibition of gene transcription (an example of epigentic silencing).   Acetylation of the tails generally leads to activation of gene transcription at that site.  Acetylation of Lys residues converts them to amides and removes the positive charge of the amine.  This would lead to decreased electrostatic interactions between the DNA and histones proteins, making the DNA more available for interaction with transcription factors and RNA polymerase.

Epigenetic changes (through methylation of DNA  or acetylation, methylation, and phosphorylation of histone proteins) causing chromatin remodeling may change phenotype (characteristics of the individual) as evidenced by the fact that identical twins can eventually diverge in ways that effect their propensities to disease.  Differences in diet and lifestyle, which can alter disease propensity, might exert their effects through epigenetic changes in gene expression.  The Human Epigenome Consortium is developing a catalog of methylation pattern differences in the human genome which might be correlated with disease risk.

The nucleosome core is about the same size as RNA polymerase.  How can RNA polymerase bind to its promoter site if it is wrapped around a nucleosome?  One obvious answer is that nucleosome are not evenly distributed on chromosomal DNA, and perhaps not even found at promoter sites on the DNA.  Rando et al. have studied the distribution of nucleosomes along the yeast genome.  They cleaved internucleosomal DNA with nucleases leaving behind the nuclease protected-DNA.  They separated the bound DNA from the nucleosome proteins, and labeled it with fluorescein.  Next, total yeast DNA was isolated, fragmented, and labeled with rhodamine.  They added both fluorescently labeled fragments to microarrays situated with overlapping 50 bp yeast chromosome 3 fragments.  Equal red and green fluorescence at a given site on the array would arise if the DNA fragments labeled with fluorescein were protected by the nucleosome protein core particle.  Low green to red fluorescence would arise if the fluorescein-labeled DNA was not protected by the nucleosome core.   

From a thermodynamic viewpoint, binding affinities for the nucleosome protein core should be the same anywhere along the chromosomal DNA.  This would lead to the prediction that nucleosomes would bind randomly along the DNA at all locations. leading to a constant ratio of green to red fluorescence across the array.  That is, there would not be district signals from the array, but rather a smeared-out signal when the DNA was extracted from many yeast cells.  The actual data showed sharp flourescein/rhodamine signals and was consistent with fact that 70% of the nucleosomes were positioned at the same position in the DNA in different cells.  Promoter sites for active genes were generally not occupied by nucleosomes.  It was unclear if these sites are always free of nucleosomes or whether protein transcription factors and RNA polymerase cause the nucleosomal core proteins to slide away from the promoter sites. 

Recent work suggests that positions of nucleosomes along the DNA is encoded in part by the DNA sequence itself, adding yet another "genetic code" that controls gene expressions.  DNA must bend around the nucleosome core.  Certain dsDNA sequences are more bendable that others, and the would be expected to have a greater chance of being involved in nucleosome complexes and less accessible for transcription.  Segal et al isolated nucleosome bound DNA sequences and developed a computation model to predict which sequence of DNA would be bendable and hence be able to easily form nucleosome complexes.  In other words, they calculated which DNA sequences would have high affinity for nucleosomes.   They concluded that 50% of the positioning of nucleosomes can be accounted for by certain DNA sequences having higher affinity of the histone octamer.    They found low nucleosome occupancy at important regulatory sites such as transcription initiation sites.    Regions of the chromosome coding for tRNA and rRNA, which are highly expressed, were found to have low nucleosome occupancy.

CONTROL OF GENE EXPRESSION BY RNA MOLECULES

What accounts for the increased complexity of organisms like humans?  As was discussed in the DNA chapter, it is not the number of chromosomes or even the number of possible genes in an organism.  One big difference between bacterial and human cells, for example, is the percentage of DNA coding for proteins.  In bacteria, most of the DNA codes for proteins, but in human eukaryotic cells, most of the DNA (up to 98%) is "junk" in that it does not code for proteins.  The DNA consists of intervening sequences within DNA coding for a given protein, and sequences between genes.  Up to 98 % of the RNA transcribed in human cells is derived from this "junk" DNA.  What function does this RNA serve?   New evidence shows that this transcribed RNA binds to other RNA molecules like mRNA (to inhibit its translation), to DNA (to control gene transcription) or to proteins (to alter gene transcription as well).  These process are called RNA interference (RNAi)

MicroRNAs (miRNAs) are formed when an enzyme called dicer cleaves a host RNA gene transcript that in animal cells contains a stem-loop hairpin structure.  Dicer recognizes the double-stranded regions of the stem.  The cleaved RNA might then bind a host mRNA, inhibiting its translation.  If the complementarity of the mRNA and miRNA is less than ideal, then binding of miRNA to the mRNA may only attenuate the translation of the mRNA.. 

Another class of RNA, short interfering or silencing RNAs (siRNAs) can also infer with mRNA translation.  siRNAs are formed when dicer cleaves viral double-stranded RNA in infected cells.  (Viruses often produced dsRNA during their life cycle).   These differ from miRNA in that siRNA are not derived by transcription from discrete host gene.  Dicer works by cleaving the dsRNA to form a small dsRNA between 20 and 25 nucleotide pairs long called siRNAs.   These can then bind to a protein complex called RISC (RNA-induced silencing complex), which promotes unraveling of the siRNA.  A complex of RISC and one of the short RNA strands from the siRNA then can bind to complementary stretches in mRNA for a specific gene (viral or host) and inhibit translation of the mRNA into protein.  Inhibition occurs when RISC complex  cleaves the RISC-siRNA- mRNA complex.   (i.e one of the components in the RISC complex is an RNA nuclease.)  This mechanism might be linked to defense mechanisms of virally-infected cells by  inhibition of viral mRNA translation. 

Jmol:  dsRNA  

When siRNAs  bind to host mRNA of complementary sequence to form a dsRNA complex, it inhibits translation of the mRNA.  This technique has recently been used to ascertain the functions of gene products in the nematode worm, C. elegans.  This organism has about 20,000 genes which code for proteins.  Kamathk et. al. have fed these worms E. Coli transformed with plasmid DNA designed to produced dsRNA upon transcription, one strand of which was complementary to mRNA sequences in the worm.  Plasmids containing almost 17,000 different dsRNA encoding genes were constructed and used to "knock out" gene expression by forming dsRNA complexes of  the mRNA with the RNAi.  Phenotypic changes in the organism were studied.  About 1700 of the dsRNA experiments led to observable (phenotypical) changes in the organism.  Genes whose inactivation was lethal (and hence were essential for survival) were generally those that had counterparts in all other organism, while those associated with nonlethal changes were more likely to be homologous to genes in higher organisms and more recently evolved.  They also selectively looked at which genes influenced lipid metabolism by incorporating a fluorescent tag which bound to lipid deposits in the organism.  Around 300 genes were found to influence fluorescence and hence regulate fat deposition in the organism. 

Figure:  RNA Interferene:  Antisense and Silencing

 

• Animation: RNAi from Imgenex

RNAi is the basis of an new emerging industry.  Many companies offer kits and free software that make RNAi studies simple.  Invitrogen is one such company. 

Figure:  RNAi-mediated gene silencing in mammals using short haripin RNA genes.
credit:  http://www.invivogen.com/sscat.php?ID=14

 

• siRNA Wizard from Invitrogen

Jmol:  Dicer 

Two groups have deleted miR-155 and looked at effects on immune cells in mice.  Immune cell function in B, T, and dendritic cells was affected, leading to animal death when exposed to salmonella after they were immunized.  Animals in sterile environments showed no effect.  In contrast to knockouts of protein-coding genes, these knockouts affect transcription of multiple genes.  Knockout of miR-208 caused heart problems in mice placed in a stressful environment.  These experiments indicated that some genetic diseases might arise from mutations in non-protein coding regions of the genome.

Recently, a new mechanism in control of gene expression has been offered which involves regulation of translation of a mRNA.  mRNA must have a sequence, the Shine-Delgarno sequence, which allows it to bind to ribosomes.  If a ligand binds to this site, mRNA could not bind to the ribosome and translation would be inhibited.  Such is the case in the mRNA encoding proteins involved in the transport and synthesis of vitamins B1 (thiamine) and B12 (adenosyl cobalamin).  Thiamin and thyamine pyrophosphate were shown to bind to the leader sequence of an E. Coli  mRNA involved in thiamine biosynthesis and inhibit the translation of the mRNA.  This allosteric mechanism for inhibition makes physiological sense since the presence  of high levels of cellular B1 would obviate the need for its synthesis or transport.  

Recognition of viral dsRNA and bacterial dsDNA by the immune system.

Before leaving the topic of RNA/proteins interactions, consider how a self cell would detect  viruses and bacteria.  It would be beneficial to the organism if the immune system could recognize and response to many types of bacteria, viruses, fungi, or protozoa by binding to common target on them. For example, it would be desirable to have a single cell type, such as a scavenging macrophage have a recognition system that would recognize a common molecular pattern such as LPS found on gram negative bacteria.  The part of the immune system that has this capability is called the innate immune system.   The cells of the innate system (dendritic cells, macrophages, eosinophils, etc) have receptors (Toll-like Receptors 1-10 or TLRs) that recognize the common pathogen associated molecular patterns - PAMPs (sometimes called MAMP - microbe associated patterns), which leads to binding, engulfment, signal transduction, maturation (differentiation), antigen presentation, and cytokine/chemokine release from these cells.  Take for example dendritic cells, which reside in the peripheral tissues and act as sentinels.  They can bind PAMPs which include:

• CHO/Lipids on bacteria surface (LPS)
• mannose (CHO found in abundance on bacteria, yeast
• dsRNA (from viruses)
• nonmethylated CpG motiffs in bacterial DNA

TLR receptors are expressed on the cell surface for recognition of external PAMPS on foreign invaders.  However, since bacterial and viral can be engulfed, it would be optimal to have intracellular recognition of viral and bacterial nucleic acids as well.  These are recognized by intracellular TLRs in the cell after the they been taken up into the cells by endocytosis.  The figure below shows how viral and bacterial nucleic acids found in endosomal vesicles, can be bound by endosomal membrane TLRs.  A Jmol model of a recent structure of TLR3 and dsRNA is shown below.

Figure:  Endosomal TLR3 Interaction with foreign RNA and DNA

 

Jmol:  TLR3:dsRNA complex 

4/29/09

Inflammation can also arise when normal tissue is damage due to injury, which exposes molecules usually located inside of the cell to the immune system.  Such molecules include high mobility group proteins (associated with chromatin), proteoglycans and nucleic acids.  These are referred to as damaged associated molecular pattern (DAMP) molecules.  Intracellular proteins exists normally in a reducing environment so when they are exposed to the oxidizing conditions of the extracellular milieu, covalent and conformational changes may ensue that

EUKARYOTIC SPECIES COMPLEXITY AND CONTROL OF GENE TRANSCRIPTION

The increasing complexity of eukaryotic organisms was thought to arise from an increasing number of genes.  This simplistic assumptions has not been validated from the results of sequencing and annotating the genomes of many eukaryotic organisms.  Compare these statistics:  the number of putative genes in the simple nematode round worm C. Elegans, the fruit fly drosophila, and the human are approximately 20,000, 14,000, and about 30,000.  There seems to be little correlation of species complexity with number of genes.  Other possible mechanisms for increasing complexity from a given genome size include producing different proteins from the same genes through differential splicing of RNA transcripts and rearranging DNA as occurs in immune cells to produce the huge repertoire of possible antibody molecules necessary for recognition of nonself molecules (such as viruses and bacteria).  These mechanisms can not account for the incredible complexity of the human species.  Levine and Tjian have proposed two other mechanisms that could account for increasing complexity.  Complexity would arise from the number of gene expression patterns and involve the involvement of nonprotein-coding regions of the genome, which in humans accounts for up to 98% of the genome.  One mechanism requires the present of greater number and complexity of DNA regulatory sequences (enhancers, silencers, promoters) in more complex organisms.  Since these sequences are in the DNA (the molecule that is transcribed), they are called cis-regulatory sequences.  The second mechanism involves an increase in the  elaboration and complexity of proteins (trans-regulatory elements) that regulate gene expression in more complex organisms..  These proteins could include transcription factors, proteins interacting with enhancer sequences, and proteins involved in chromatin remodeling (described above).  They estimate that up to a third of the human genome (1 billion base pairs) might be involved in the regulation of gene transcription.  In addition, 5-10% of all proteins expressed from genes appear to regulate gene transcription.  There appears to be about  300, 1000, and 3000  transcription factor in yeast, drosophila and C. elegans, and humans, respectively.  There is about one transcription factor for every gene in yeast, but one for every ten in humans. 

In simple eukaryotes, cis regulatory elements would include the promoter (TATA box region), and upstream regulatory sequences (enhancer) and silencers about 100-200 base pairs from the promoter.  In more complex eukaryotic species like humans,  the promoter is more complex, containing the TATA box, initiator sequences (INR) and downstream promoter elements (DPE).  Upstream cis regulatory elements (as far as 10 kb from the promoter) include multiple enhancers, silencers, and insulators.  Most promoters have TATA boxes, where TATA Binding Protein (TBP) binds.  Upstreams elements in turn regulate the binding of TBP. 

• Eukaryotic promoters and regulatory regions
• Eukaryotic multisubunit general transcription apparatus
• Biological Regulation: BioBase - Gene Regulation (TRANS-FAC 6.0 public site free with registration)

Comparative Genomics - Gene Expression Differences Between Humans and Chimpanzees

Our closest biological relative is the chimpanzee, who branched off from a common ancestor of both of us about six million years ago.  Our DNA sequence appears to be 98.6 % identical (not just homologous).  If we are so close in our genetic blue print, how can we be so different.  There are many possible conjectures that can be answered by comparing the chimp and human genomes.  Our genes are presumably very similar.  People suspect that there are two major kinds of differences that make our species different:

• our genes are very similar but are transcribed differently in the two species.  Recent evidence show that the types of RNA transcribed by human and chimp livers are very similar, but many more genes are transcribed in human brains compared to chimp brains.
• humans may have lost genes (or their function) that are required for chimp survival in the jungle.  The observations that chimps are resistant to many of the disease pathogens  that affect humans (immunodeficiency viruses like HIV, influenza A virus, hepatitis B/C, malarial parasite)  could be explained by the loss of "protective" genes in humans.  In addition, cardiovascular disease and certain types of cancer are rarer in chimps.  Humans have apparently  "lost" genes involved in body hair, strength, and early maturation, traits that would adapt the chimp to life in the jungle. 

We previously discussed an example of a loss of gene function in humans.  We have lost a hydroxylase gene involved in formation of certain types of sialic acids, specifically N-glycolylneuraminic acid, found on cell surface glycogroteins of mammals other than humans. Chimps have a lectin receptor for this sialic acid.  Recent work has shown that human lack a critical Arg in our version of the lectin that would recognize N-glycolylneuraminic acid, making it unable to bind this ligand.  Hence both pairs of genes involved in these type of interactions (cell:cell) are missing.  Since sialic acid molecules are often involved in pathogen:host binding, these difference in humans compared to chimps might account for the difference in disease susceptibility as mentioned above.

With respect to gene transcription in the brain, Lai et al. have found a mutation in the human gene FOXP2, a transcription factor, in a family that has significant difficulty in controlling muscles required for articulation of words. This mutation also causes problems in language processing and grammar construction.  Comparison of the normal human gene with other primate genes shows distinct differences in the human gene which may have conferred on human the ability to use speech.

Chimp chromo 22 is homologous to human chromosome 21.  Recently, sequencers have found 1.44% single nucleotide changes between the two, a finding in line with overall homology between chimp and human DNA of 98.6%.  The surprising finding was 68,000 insertions and deletions (indels) compare to humans.  Most were short (<30 nucleotides).  Those longer than 300 involved mobile genetic elements (transposons). Humans have a much higher incidence of insertions called Alu repeats.  A high figure of 20% of homologous genes displayed significantly different expression levels. 

In September 2005, a draft sequence of the chimpanzee genome and a comparison with the human genome was published by The Chimpanzee Sequencing and Analysis Consortium.  Here are some of their findings:

• "single nucleotide substitutions occur at a mean rate of 1.23% between copies of the human and chimpanzee genome."
• "insertion and deletion (indel) events are fewer in number than single-nucleotide substitutions, but result in 1.5% of the euchromatic sequence in each species being lineage-specific."
• "There are notable differences in the rate of transposable element insertions: short interspersed elements (SINEs) have been threefold more active in humans, whereas chimpanzees have acquired two new families of retroviral elements."
• "Orthologous proteins in human and chimpanzee are extremely similar, with ~29% being identical and the typical orthologue differing by only two amino acids."

Since their genomes are over 3 billion base pairs, a 2% difference would mean around 60 million differences.  The actual number appears to be 35 million single nucleotide differences (not counting insertions and deletions).  Most of these would be expected not to be in genes and have little overall effect on phenotypic differences between the species.  Finding the critical difference will be time consuming, and may requiring the sequencing of other primate genomes.

In their summary of the finding, Li and Saunders discuss changes in nucleotides that are synonymous (no changes in amino acids in the protein) and nonsynonymous.  If a region of a gene can not tolerate changes that lead to amino acid alterations (i.e the nucleotides are under significant selective pressure not to change), the nonsyonymous rate of substitution would be lower than the rate of synonymous change.  If change can occur without structure/function loss in the protein, the two rates would be similar.  Comparing over 13,000 gene pairs from both organisms, they found the nonsynonymous rate to be about 25% of the synonymous rate.   Hence most of the genes are conserved between species and would not be expected to contribute to the phenotypic difference in the organisms.  Of the genes that showed higher nonsynonymous rates, none were obviously linked to brain function, but many were involved in immune function. 

The biggest differences between the genomes were insertions/deletions (indels, numbering around 5 million) and gene duplications, not single nucleotide mutation.  Insertions are often of two classes.  Insertions include duplication of DNA stretches and addition of transposons ("jumping" gene or moveable DNA elements).  These can be small (such as Alu repeats) or long (such as L1 insertions).  In the human genome there are 7000 Alu sequences but in chimps there are 2300.  Both have about equivalent numbers of Li insertions.  Given that we have lost some traits (such as hair and strength), perhaps some chimp genes were lost in the human genome by the presence of indels.  53 such genes human genes were found.  Perhaps the biggest change between chimps and humans is altered gene expression, which was not studied in this paper.

In another study by Xiaoxia Wang et al, a comparison was made of "pseuodgene" in humans (genes that acquired mutations in the past that disrupted their expression as functional protein and compared them to corresponding genes in chimps that still maintain function (i.e. they lead to functional proteins).  Analysis showed that the identified pseudogenes were not randomly distributed among different classes of genes.  Rather, there were concentrated in genes encoding olfactory receptor proteins, bitter tastant receptors, and immune system genes.  Homo sapiens have a much diminished sense of smell.  Bitter receptors probably became less important as humans switched from plants which contain many bitter toxins to meats.  They attribute changes in immune system genes to changes in environment which might lead to gene lose if the intensity of the immune response, and the balance of immune self and nonself recognition might be altered in different environmental conditions. 

 

Another major difference has been noted in gene copy number.   Work by Hahn et al shows that gene copy number between human and chimps differ by 6.4%.  After diverging from a common ancestor, humans gained 689 copies of some genes, compared to 26 for chimps.  Likewise, humans lost 86 copies of some genes compare to a lose in chimps of 729 copies. 

What maintains species?  Barriers to interspecies hybrids.

New species seem to arise, according to evolutionary theory, when members of a species become geographically isolated.  Each separated population accrues different mutations in their genomes, which confer adaptive advantages to each population in their different environment.  With a long enough divergence time, genetic barriers to the production of viable hybrids between the population develop, leading to the divergence of the populations into separate species.  This rational explanation doesn't give a specific molecular mechanism causing hybrid failure.   In the 1930s, Dobzhansky and Muller proposed that changes in two genes that produce proteins that interact could account for interspecies hybrid failure.  These genes would presumably mutate at a faster rate than usual.  Within a species, the two genes would co-mutate at similar rates to produce proteins that still interact, but fast evolutionary change in the others "soon to be new species" gene pair would make hybrids produced from mating infertile at best, or lethal.  

Brideau et al. have found a gene pair, lethal hybrid rescue (Lhr), that in Drosophila simulans diverged functionally, and hybrid male rescue (Hmr) in Drosophila melanogaster, which also has diverged functionally.  F1 hybrid male offspring from crosses died. The Hmr gene in D. melanogaster is a transcription factor.  The hmr gene is one of the most rapidly evolving genes in the genome.  The exact function of the Lhr gene is uncertain but is associated with condensed chromatin (heterochromatin). 

 

Pre-Class Questions:    Binding: D. Transcripton - Question

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Moodle Online Quiz (PASSWORD PROTECTED):  TRANSCRIPTION

Sliding Model for protein/DNA Interactions

Recent References

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