control gene expression

CHAPTER 6

Copyright © 2013 John Wiley & Sons, Inc. All rights reserved.

Controlling Gene Expression

Keys

© 2013 John Wiley & Sons, Inc. All rights reserved.

Describe the fine structure of the nucleus and the nuclear envelope. Outline the levels of structure of chromatin and chromosomes. Describe the composition of nucleosomes; importance of histones to the structure. Distinguish between the structure/functions of heterochromatin and chromatin. Distinguish between the functions of facultative and constitutive chromatin. Discuss purposes of centromeres/telomeres to chromosome structure and function. Emphasize the high degree of organization now apparent in the nucleus. Outline the control of gene expression in prokaryotes using the lac and trp operons. Distinguish between the concepts of positive and negative control of gene

expression. Emphasize importance of transcription factors to gene expression control in

eukaryotes. Describe common features of transcription factor structure; DNA-binding sites. Discuss strategies used by eukaryotes to regulate gene expression. Explain the influence of DNA methylation on eukaryotic gene expression.

Introduction


All cells in a multi-cellular organism contain the same complement of genes.

Cells express their genetic information selectively.

Gene expression is controlled by regulatory machinery in the cell nucleus.

(6.1) Control of Gene Expression in Bacteria


Bacterial cells selectively express genes to use the available resources effectively. The presence of

lactose in the medium indices the synthesis of the enzyme β-galactosidase.

The presence of tryptophan in the medium represses the genes that encode enzymes for tryptophan synthesis. The kinetics of β-galactosidase induction

in E. coli: mRNA and protein induction


The Bacterial Operon An operon is a functional complex of genes containing the information for

enzymes of a metabolic pathway. It includes: Structural genes – code for the enzymes and are translated from a

single mRNA that is usually polycistronic (encodes for more than one protein).

Promoter – where the RNA polymerase binds. Operator – site next to promoter where the regulatory protein can bind.

Control of Gene Expression in BacteriaThe Bacterial Operon

Organization of a bacterial operon. Enzymes in a metabolic pathway are encoded by a series of structural genes that reside in

a contiguous array within the bacterial chromosome.


The Bacterial Operon The key to operon expression lies in the repressor.

A repressor which binds to a specific DNA sequence to determine whether or not a particular gene is transcribed. RNA polymerase is unable to bind to the promoter if the repressor is bound.

The regulatory gene encodes the repressor protein.

Control of Gene Expression in BacteriaThe Bacterial Operon

Organization of a bacterial operon. Enzymes in a metabolic pathway are encoded by a series of structural genes that reside in

a contiguous array within the bacterial chromosome.

Gene regulation by operons


The lac Operon It is an inducible

operon, which is turned on in the presence of lactose (inducer).

The lac operon contains three structural genes.

Lactose binds to the repressor, changing its conformation and making it unable to bind to the operator.

A repressor protein can bind to the operator and prevent transcription in the absence of lactose.


The lac Operon: catabolite repression The lac repressor exerts negative control. The glucose effect is an example of positive control. Cyclic AMP (cAMP) acts by binding to a cAMP receptor protein (CRP). Binding of CRP-cAMP to the lac control region changes the

conformation of DNA thus allowing RNA polymerase to transcribe the lac operon.

Control of Gene Expression in BacteriaThe lac Operon

Nucleotide sequence of binding sites in the control region of the lac operon

Gene regulation by operons


The trp Operon It is a repressible

operon, which is turned off in the presence of tryptophan.

The trp operon repressor is active only when it is bound to a corepressor such as tryptophan.


Riboswitches A number of bacterial mRNAs can bind to a small metabolite in their 5’

untranslated region, which in turn alters the gene involved in the production of such metabolite.

These mRNAs are called riboswitches because they undergo a conformational change and can suppress gene expression.

Most riboswitches suppress gene expression by blocking either termination of transcription or initiation of translation.

Riboswitches allow bacteria to regulate gene expression in response to some metabolites.

Given that they act without the participation of protein cofactors, riboswitches are likely another legacy from an ancestral RNA world.

Control of Gene Expression in BacteriaRiboswitches

(6.2) Control of Gene Expression in Eukaryotes: Structure and Function of the Cell Nucleus


The contents of the nucleus are enclosed by the nuclear envelope.

A typical nondividing nucleus includes: Chromosomes as extended fibers of chromatin. Nucleoli for rRNA synthesis. Nucleoplasm as the fluid where solutes are dissolved. The nuclear matrix, which is the protein-containing fibrillar network.

The cell nucleus. EM of an interphase

HeLa cell nucleus (left) and schematic

drawing of major components (right).

The nuclear envelope. Schematic drawing (top) and EM of the nuclear envelope of

an onion root tip cell (bottom)

Control of Gene Expression in EukaryotesThe nuclear envelope


The Nuclear Envelope The nuclear envelope is a

structure that divides the nucleus from its cytoplasm.

It consists of two membranes separated by a nuclear space.

The two membranes are fuses at sites forming a nuclear pore.

The inner surface of the nuclear envelope is lined by the nuclear lamina.

Contains around 60 distinct transmembrane proteins.

Control of Gene Expression in EukaryotesThe nuclear lamina


The nuclear lamina Supports the nuclear envelope and it is composed of lamins. Integrity of nuclear lamina regulated by

phosphorylation/dephosphorylation. Human conditions: lamin A/C mutation gives Hutchinson-Gilford Progeria

syndrome, lamin B mutation causes leukodystrophy (loss of myelin)

Nucleus stained for nuclear lamina (R) and

nuclear matrix (G)

EM: metal-shadowed nuclear envelope of a

Xenopus oocyte

Micrographs of fibroblast nuclei from a patient with HGPS (bottom)

or a healthy subject (top).

Control of Gene Expression in EukaryotesThe nuclear pore complex


Structure of Nuclear Pore Complex and its Role in Nucleocytoplasmic Trafficking Proteins and RNA are transported

in and out of the nucleus. Nuclear pores contain the nuclear

pore complex (NPC) that appears to fill the pore like a stopper.

NPC is composed of ~30 proteins called nucleoporins.

Movement of materials through the nuclear pore: EM (frog oocyte) after injection with gold particles coated with nuclear protein (left, middle) and EM of an insect cell showing the movement of granular material (ribosomal subunit, right)

Control of Gene Expression in Eukaryotes The nuclear pore complex


Structure of Nuclear Pore Complex and its Role in Nucleocytoplasmic Trafficking Proteins and RNA are transported in and out of the

nucleus. Nuclear pores contain the nuclear pore complex

(NPC) that appears to fill the pore like a stopper. NPC is composed of ~30 proteins called

nucleoporins.

Scanning electron micrographs of the nuclear pore complex from an amphibian oocyte. Cytoplasmic (left) and nuclear (middle) faces of the nuclear envelope complex. Nuclear face shows NPC distribution and intact patches of the nuclear lamina (NEL, right).



Structure of Nuclear Pore Complex and its Role in Nucleocytoplasmic Trafficking

Huge complex (15-30X mass of a ribosome) that exhibits octagonal symmetry. Channel: 20-to 30-nm-wide FG (phenylalanine-glycine) domains form a hydrophobic sieve that blocks the

diffusion of larger macromolecules (greater than about 40,000 Daltons).

Model of a vertebrate nuclear pore complex (NPC). The structure

consists of several parts, including a scaffold that anchors the complex to the nuclear envelope, a cytoplasmic and a nuclear ring, a nuclear basket,

and eight cytoplasmic filaments.



Structure of Nuclear Pore Complex and its Role in Nucleocytoplasmic Trafficking

Huge complex (15-30X mass of a ribosome) that exhibits octagonal symmetry. Channel: 20-to 30-nm-wide FG (phenylalanine-glycine) domains form a hydrophobic sieve that blocks the

diffusion of larger macromolecules (greater than about 40,000 Daltons).

Model of a vertebrate nuclear pore complex (NPC). Three dimensional reconstruction of a portion of a nuclear pore complex showing the localization of individual nucleoporin molecules within the structure

Control of Gene Expression in Eukaryotes Importing proteins


Proteins synthesized in the cytoplasm are targeted for the nucleus by the nuclear localization signal (NLS), e.g. P-K-K-K-R-K-V having basic residues. Proteins with an NLS stretch bind to an NLS receptor (importin). Conformation of the NPC changes as the protein passes through. RNAs move through NPCs as RNPs and carry NES (nuclear export signals)

to pass.

Importing proteins into the nucleus. Steps in nuclear protein import (left). Gold particle-nucleoplasmin injection into frog oocytes shows binding to cytoplasmic filaments (right)

Control of Gene Expression in EukaryotesChromosomes and Chromatin


Chromosomes and Chromatin Packaging the Genome

Chromosomes consist of chromatin fibers, composed of DNA and associated proteins.

Each chromosome contains a single, continuous DNA molecule.

Nucleosomes: The Lowest Level of Chromosome Organization The protein component of

chromosomes include histones, a group of highly conserved proteins.

Histones have a high content of basic amino acids.



DNA and histones are organized into repeating subunits called nucleosomes.

Each nucleosome includes a core particle of supercoiled DNA and histone H1 serving as a linker.

DNA is wrapped around the core complex.

The histone core complex consists of two molecules each of H2A, H2B, H3, and H4 forming an octamer.

H3H4H2B

H2A

H3H2B

Nucleosomal organization of

chromatin: Schematic diagram

(top) and EM of Drosophila cell nucleus with

nucleosomes along DNA strand

(bottom)



DNA and histones are organized into repeating subunits called nucleosomes.

Each nucleosome includes a core particle of supercoiled DNA and histone H1 serving as a linker.

DNA is wrapped around the core complex.

The histone core complex consists of two molecules each of H2A, H2B, H3, and H4 forming an octamer.

3D structure of a nucleosome from X-ray

crystallography. Core particle at two views (top) and schematic of half of a

core particle (side)


Histone modification is one mechanism to alter the character of nucleosomes.

DNA and histones are held together by noncovalent bonds. Ionic bonds between negatively charged phosphates of the DNA

backbone and positively charged residues of the histones. Histones, regulatory proteins, and enzymes dynamically mediate

DNA transcription, compaction, replication, recombination, and repair.


Control of Gene Expression in EukaryotesHigher Levels of Chromatin Structure


Higher Levels of Chromatin Structure A 30-nm filament is

another level of chromatin packaging, maintained by histone H1.

Chromatin filaments are organized into large supercoiled loops.

The presence of loops in chromatin can be seen: In mitotic chromosomes

form which histones have been extracted.

In meiotic lampbrush chromosomes from amphibian oocytes.

30-nm fiber: EM of a fiber (left) and two packaging models (middle, right).



Higher Levels of Chromatin Structure A 30-nm filament is

another level of chromatin packaging, maintained by histone H1.

Chromatin filaments are organized into large supercoiled loops.

The presence of loops in chromatin can be seen: In mitotic chromosomes

form which histones have been extracted.

In meiotic lampbrush chromosomes from amphibian oocytes.

Chromatin loops: a higher level of chromatin structure. EM: of a

mitotic chromosome (left) and model for cohesin in maintaining

loops (right)



Higher Levels of Chromatin Structure A nucleus 10 m in

diameter can pack 200,000 times this length of DNA within its boundaries.

Packing ratio of the DNA in nucleosomes is approximately 7:1.

Assembly of the 30-nm fiber increases the DNA-packing ratio to 40:1.

Mitotic chromosomes represent the ultimate in chromatin compactness with a ratio of 10,000:1.

Levels of organization of chromatin.

Control of Gene Expression in EukaryotesHeterochromatin and Euchromatin


Heterochromatin and Euchromatin Euchromatin returns to a dispersed state after mitosis. Heterochromatin is condensed during interphase.

Constitutive heterochromatin remains condensed all the time. Found mostly around centromeres and telomeres. Consists of highly repeated sequences and few genes.

Facultative heterochromatin is inactivated during certain phases of the organism’s life. Is found in one of the X chromosomes as a Barr body

through X inactivation. X inactivation is a random process, making adult females

genetic mosaics.

Calico cat cloning: Random inactivation of the X chromosome in different cells

during embryonic development creates a mosaic of tissue patches.

Control of Gene Expression in Eukaryotes Heterochromatin and Euchromatin


• Facultative heterochromatin is inactivated during certain phases of the organism’s life.

– Is found in one of the X chromosomes as a Barr body through X inactivation.– X inactivation is a random process, making adult females genetic mosaics.

Inactivated X chromosome or Barr body (arrows).

Control of Gene Expression in EukaryotesThe histone code


The Histone Code and Formation of Heterochromatin The histone code hypothesis states that the activity of a chromatin

region depends on the degree of chemical modification of histone tails.

Histone tail modifications influence chromatin in two ways: Serve as docking sites to recruit nonhistone proteins. Alter the way histones of neighboring nucleosomes interact.

The “histone code.” Histones can be

modified by addition of methyl, acetyl, and

phosphate groups

Control of Gene Expression in EukaryotesThe histone code


The majority of modified amino acids reside on the N-termini of H3 and H4. Each of the bound proteins possesses an activity that alters the structure and/or function of the chromatin. Heterochromatin has many methylated H3 histones, which stabilize the compact nature of the chromatin. Small RNAs and specific enzymes play a role in histone methylation.

Proteins that bind selectively to modified H3 or H4 residues

Correlation between transcriptional activity and histone acetylation. Chromosomes labeled with fluorescent antibodies to acetylated histone H4 stain all chromosomes except the inactivated X (arrow).


Removal of the acetyl groups from H3 and H4 histones is among the initial steps in conversion of euchromatin into heterochromatin.

Histone deacetylation is accompanied by methylation of H3K9 histone methyltransferase (SUV39H1 in humans.

Methylated H3K9 binds to proteins with a chromodomain, for example heterochromatic protein 1 (HP1)

Once HP1 is bound to the histone tails, HP1-HP1 interactions facilitate chromatin packaging into a heterochromatin state,

Control of Gene Expression in EukaryotesHistone modification

Control of Gene Expression in Eukaryotes

Histone modification


Removal of the acetyl groups from H3 and H4 histones is among the initial steps in conversion of euchromatin into heterochromatin.

Histone deacetylation is accompanied by methylation of H3K9 histone methyltransferase (SUV39H1 in humans.

Methylated H3K9 binds to proteins with a chromodomain, for example heterochromatic protein 1 (HP1)

Once HP1 is bound to the histone tails, HP1-HP1 interactions facilitate chromatin packaging into a heterochromatin state,

Model of possible events during the

formation of heterochromatin

Histone deacetylaseHistone methyltransferase


The Structure of a Mitotic Chromosome Chromatin of a mitotic cell exists

in its most highly condensed state. Staining mitotic chromosomes can

provide useful information. A karyotype is a preparation of

homologous pairs ordered according to size.

The pattern on a karyotype may be used to screen chromosomal abnormalities.

Control of Gene Expression in EukaryotesThe Structure of a Mitotic Chromosome

Procedure to prepare mitotic chromosomes

for microscopic observation

from leukocytes


The Structure of a Mitotic Chromosome Chromatin of a mitotic cell exists

in its most highly condensed state. Staining mitotic chromosomes can

provide useful information. A karyotype is a preparation of

homologous pairs ordered according to size.

The pattern on a karyotype may be used to screen chromosomal abnormalities.

Control of Gene Expression in EukaryotesThe Structure of a Mitotic Chromosome

Human mitotic chromosomes labeled with

different specific fluorescent dyes.

The stained chromosomes of

a human male arranged in a

karyotype


Telomeres The end of each chromosome is called a telomere

and is distinguished by a set of repeated sequences. New repeats are added by a telomerase, a reverse

transcriptase that synthesizes DNA from a DNA template.

Telomeres are required for the complete replication of the chromosome because they protect the ends from being degraded.

Telomerase activity is thought to have major effects on cell life.

Control of Gene Expression in EukaryotesTelomeres

In situ hybridization with a DNA

probe (TTAGGG) to

locate telomeres on

human chromosome

Proteins can bind to

telomeres: RAP1 in

yellow, DNA in blue








The end-replication problem: Generation of single stranded overhangs that shorten DNA







Control of Gene Expression in Eukaryotes Telomeres

The single-stranded overhang is not free but forms a loop. The loop is a binding site for telomere-capping proteins that protect the ends of the chromosomes

and regulate telomere length.


Telomeres The end of each chromosome is called a telomere and is

distinguished by a set of repeated sequences. New repeats are added by a telomerase, a reverse

transcriptase that synthesizes DNA from a DNA template. Telomeres are required for the complete replication of

the chromosome because they protect the ends from being degraded.



The mechanism of action of telomerase. Gap in complementary strand

filled by DNA polymerase (carries DNA primer).


Telomeres The end of each chromosome is called a telomere and is

distinguished by a set of repeated sequences. New repeats are added by a telomerase, a reverse

transcriptase that synthesizes DNA from a DNA template. Telomeres are required for the complete replication of

the chromosome because they protect the ends from being degraded.


Control of Gene Expression in Eukaryotes Telomeres

The importance of telomerase in maintaining chromosome integrity.

Chromosomes from a telomerase knockout mouse cell shows some

chromosomes lack telomeres entirely (stained yellow) and some have fused

to one another at their ends


Telomeres In somatic cells, telomere lengths are reduced each cell division

to limit cell doublings. A critical point occurs from telomere shortening when cells stop

their growth and division. In contrast, cells that are able to resume telomerase expression

continue to proliferate. These cells continue to divide and do not shown normal signs of

aging. Approximately 90% of human tumors have cells with active

telomerase.


Telomerase dynamics during normal and abnormal growth. Limited

telomerase levels in somatic cells reduces the amount of cell doublings

compared to germ cells, unless telomerase is reactivated.


Centromeres The centromere is located at the site markedly indented on a chromosome. Centromeres contain constitutive heterochromatin. Centromeric DNA is the site of microtubule attachment during mitosis. DNA sequence is not important for centromere structure and function. Histone H3 variant CENP-A is found in the centromeres to potentially function in

kinetochore assembly.

Control of Gene Expression in EukaryotesCentromeres

Scanning electron micrograph of a mitotic chromosome with the

centromere marked by a distinct indentation.


Epigenetics: There’s More to Inheritance than DNA Epigenetic inheritance depends on factors other than DNA sequences. X-chromosome inactivation is an example, since the two X chromosomes can have identical DNA sequences, but one is inactivated and the other is not. An epigenetic state can usually be reversed; X chromosomes, for example, are reactivated prior to formation of gametes. Differences in disease susceptibility and longevity between genetically identical twins may be due, in part, to epigenetic differences that appear

between the twins as they age. Parental histones determine the chemical modifications found in the newly synthesized histones. As heterochromatin is replicated, a histone methyltransferase labels the newly synthesized H3 molecules added into the daughter nucleosomes. Euchromatic regions tend to contain acetylated H3 tails, a modification transmitted from parental chromatin to progeny chromatin.

Control of Gene Expression in EukaryotesEpigenetics


The Nucleus as an Organized Organelle Chromatin fibers of an interphase chromosome are not diffuse and

random, but are concentrated into distinct territories. Genes are physically moved to nuclear sites called transcription factories

where transcription machinery is located (e.g., hormone induction). DNA sequences that participate in a common biological response but

reside on different chromosomes interact within the nucleus.

Control of Gene Expression in EukaryotesNuclear organization

3D map of all of the chromosomes present in a human fibroblast nucleus. Each chromosome,

represented as an identifiable color, is found to occupy a distinct territory within the nucleus.


Localizing specific chromosomes within an interphase nucleus. More

active chromosomes, those that have more protein-coding genes,

are centrally located in the nucleus.




where transcription machinery is located (e.g., hormone induction). DNA sequences that participate in a common biological response but


Control of Gene Expression in Eukaryotes Nuclear organization

Breast cancer cells treated

with estrogen co-activate

genes on Chr2 and Chr21

Model of how different DNA regions could be organized

for gene expression




where transcription machinery is located (e.g. hormone induction). DNA sequences that participate in a common biological response but



Antibody staining against an mRNA processing factor

shows 30-50 distinct sites Time course of viral gene expression in

infected cells showing splicing factors (orange) compared to integration site (white arrow)




where transcription machinery is located (e.g. hormone induction). DNA sequences that participate in a common biological response but


The Human Perspective: Chromosomal Aberrations and Human Disorders


A chromosomal aberration is loss or exchange of a segment between different chromosomes, caused by exposure to DNA-damaging agents.

Chromosomal aberrations have different consequences depending on whether they are in somatic or germ cells. Inversions involve the breakage of a chromosome and

resealing of the segment in a reverse order. Translocations are the result of the attachment of all or

one piece of one chromosome to another chromosome.

The effect of inversion. Crossing

over between a normal chromosome

(purple) and one containing an

inversion (green)

Translocation. Exchange between chr12 (bright blue) and chr7 (red) in

human cells

The Human Perspective: Chromosomal Aberrations and Human Disorders


A chromosomal aberration is loss or exchange of a segment between different chromosomes, caused by exposure to DNA-damaging agents.

Chromosomal aberrations have different consequences depending on whether they are in somatic or germ cells. Deletions result when there is loss of a portion of a

chromosome. Duplications occur when a portion of a chromosome

is repeated.

Translocation and evolution. If the only two ape chromosomes that have no counterpart in humans are hypothetically fused, they match

human chromosome number 2, band for band.

(6.3) An Overview of Gene Regulation in Eukaryotes


Cells of a complex eukaryote exist in many differentiated states. Differentiated cells

retain a full set of genes.

Nuclei from cells of adult animals are capable of supporting the development of anew individual, as demonstrated in experiments.

Cloning of animals demonstrates that

nuclei retain a complete set of

genetic information

An Overview of Gene Regulation in Eukaryotes


Genes are turned on and off as a result of interaction with regulatory proteins. Each cell type contains

a unique set of proteins. Regulation of gene

expression occurs on three levels: Transcriptional

control Processing control Translational control Posttranslational

controlOverview of levels of

control of gene expression

(6.4) Transcriptional control


Differential transcription is the most important mechanism by which eukaryotic cells determine which proteins are synthesized.

Differential gene expression is found in various condition: Cells at different stages of

embryonic development Cells in different tissues Cells that are exposed to different

types of stimuli

Experimental demonstration of the tissue-specific expression of a

gene involved in muscle cell differentiation. A transgenic

mouse embryo that contains the regulatory region of the myogenin

gene placed upstream from a bacterial β-galactosidase gene,

which acts as a reporter.


DNA microarrays can monitor the expression of thousands of genes simultaneously. Immobilized fragments of

DNA are hybridized with fluorescent cDNAs.

Genes that are expressed show up as fluorescent spots on immobilized genes.

Microarrays a provide a visual picture of gene expression.

Transcriptional control DNA microarrays

The construction of DNA microarrays


DNA microarrays can monitor the expression of thousands of genes simultaneously. Immobilized fragments of DNA are hybridized with fluorescent cDNAs. Genes expressed show up as fluorescent spots on immobilized genes. Microarrays a provide a visual picture of gene expression.


Experimental results comparing yeast in glucose or ethanol

Plot showing changes in glucose and ethanol concentrations in the

media and in cell density

Changes in expression of genes for TCA-cycle

enzymes


DNA microarrays can facilitate the diagnosis and treatment of human diseases. Human breast tumors can be ER-positive or ER-negative. These tumor subtypes show differences in a subset of

genes. Personalized medicine in the future will be reliant upon

transcription profiling, not only for the diagnosis and to establish a treatment plan, but also to monitor the effectiveness of the treatment.


Transcription profiling to personalize breast cancer therapy. ER+ and ER- tumors show 179

genes that are differentially regulated. Expression ranges from low (blue) to high (red).


The Role of Transcription Factors in Regulating Gene Expression Transcription factors

are the proteins that either acts as transcription activators or transcription inhibitors. A single gene can be

controlled by different regulatory proteins.

A single DNA-binding protein may control the expression of many different genes.

Transcriptional control Transcription factors

Combinatorial control of transcription. Transcription of the Oct4 gene requires the action of multiple transcription factors that

bind upstream of the start site of transcription.

Interactions between transcription factors bound to different gene regions. Image of two separate transcription factors, NFAT-1 (green) and AP-1 (red and blue) bound to DNA.


The Role of Transcription Factors in Regulating Gene Expression Transcription factors

are the proteins that either acts as transcription activators or transcription inhibitors. A single gene can be

controlled by different regulatory proteins.

A single DNA-binding protein may control the expression of many different genes.


Phenotypic conversion induced by abnormal expression of a single transcription factor. The leg of this fruit fly bears a fully formed eye that has developed due to the forced expression of the eyeless gene


The Role of Transcription Factors in Regulating Gene Expression Gene expression gives

rise to phenotypes within cells and tissues of an organism. Ectopic or forced gene

expression can alter the phenotype.

Expression of MyoD causes fibroblasts to become muscle cells.

Expression of eyeless can trigger eye formation in the leg of flies.



The Role of Transcription Factors in Determining a Cell’s Phenotype Embryonic stem (ES) cells are:

Capable of indefinite self-renewal Pluripotent, capable of differentiating into all of the different types of

cells. The importance of transcription factors in ES cells was demonstrated

when these factors were introduced and shown to reprogram these cells.

Introducing a combination of genes encoding only four specific transcription factors (Oct4, Sox2, Myc, and Klf4) was sufficient to reprogram the fibroblasts and convert them into undifferentiated cells that behaved like ES cells.

The induced pluripotent cells, or iPS cells, that were generated in these early experiments were capable of dividing indefinitely in culture and of differentiating into all of the various types of the body’s cells.



The Structure of Transcription Factors Transcription factors contain a

DNA-binding domain and an activation domain.

Many transcription factors can bind a protein of identical or similar structure to form a dimer.

Transcription Factor Motifs The DNA-binding domains of

most transcription factors have related structures (motifs) that interact with DNA sequences.

Most motifs contain a segment that binds to the major groove of the DNA.

Transcriptional controlTranscription factors

Interaction between dimeric glucocorticoid receptor (GR) and DNA, with zinc ion co-factor (purple spheres)


Transcription factor motifs The zinc finger motif –

the zinc ion of each finger is held in place by two cysteines and two histidines.

The helix-loop-helix (HLH) motif – has two α-helical segments separated by a loop.

The leucine zipper motif – has a leucine at every seventh amino acid of an α-helix.


Complex between GLI (has five zinc fingers) and DNA.

Each fingers is colored differently. Inset: structure of a single zinc finger.

A model of TFIIIA bound to the DNA of the 5S RNA gene







MyoD is a dimeric bHLH transcription factor for muscle cell differentiation. (Left): Basic (red) and HLH regions (brown) are shown.

The DNA bases bound are indicated (yellow). (Right): Sketch of the MyoD complex

AP-1FosJun







AP-1, a bZIP transcription factor, is a heterodimer between Fos

(red) and Jun (blue) that plays a role in cell proliferation


bHLH and HLH-containing transcription factors play a key role in the differentiation of certain tissues.

bHLH and HLH-containing transcription factors also participate in the control of cell proliferation and cancer.

Heterodimerization greatly expands the diversity of regulatory factors that can be generated from a limited number of polypeptides


Increasing the DNA-binding specificities of transcription factors through dimerization The human genome encodes approximately 118 different

bHLH monomers


The Glucocorticoid Receptor: An Example of Transcriptional Activation Various hormones that affect the expression of the PEPCK gene are insulin, thyroid

hormone, glucagon, and glucocorticoids. These hormones act by means of specific transcription factors that bind to the DNA PEPCK is a key enzyme controlled by a variety of transcription factors called response

elements.

Transcriptional control DNA binding sites

Regulating transcription from the rat PEPCK gene.

Transcription is controlled by a many transcription factors

binding DNA regulatory sequences upstream from the

gene’s coding region


DNA Sites Involved in Regulating Transcription The TATA box regulates the initiation of transcription. The core promoter, from the TATA box to the start, is where the

initiation complex assembles. The CAAT and the GC box are upstream and are required for initiation. Alternative promoters allow some genes to be transcribed at more

than one site.


Identifying promoter sequences required for transcription. Deletion

experiments of the PEPCK gene promoter


Researchers use the following techniques to find DNA sequences involved in regulation: Deletion mapping DNA footprinting Genome-wide

location analysis Allows simultaneous

monitoring of all the sites within the genome that carry a particular activity.

Chromatin immuno-precipitation (ChIP)


ChIP: global search for

transcription-factor binding

sites


The Glucocorticoid Receptor The glucocorticoid receptor (GR) is a nuclear receptor that includes a ligand-

binding domain and a DNA-binding transcription factor. The GR binds to a glucocorticoid response element (GRE), which is a

palindrome. A palindrome is when the two DNA strands have the same 5 to 3 sequence.


Steps in gene activation by the steroid hormone cortisol via the glucocorticoid

receptor


Transcriptional Activation: The Role of Enhancers, Promoters, and Coactivators Enhancers are DNA elements that stimulate transcription.

Can be located very far upstream from the regulated gene. A promoter and its enhancers can be “cordoned off” from other

elements by sequences called insulators.

Transcriptional control Transcriptional activation

A survey of the means by which transcriptional

activators bound at distant sites can influence gene

expression


Coactivators serve as intermediates for transcription factors, and are divided into two classes: Those that interact with

the transcription machinery.

Those that alter chromatin structure modifying histones to regulate transcription. By using histone

acetyltransferases (HATS)

By using chromatin remodeling complexes


Location of histone modifications in the chromatin of transcribed genes (yeast). Acetylation occurs usually in active gene

promoters and decreases in the transcribed portion, whereas H3K36 methylation has the reverse pattern. TSS, transcription start site.

Model of events following the binding of a transcriptional activator















Alternative actions of chromatin remodeling complexes: ‘sliding’ exposes TATA site;

‘conformational change’ exposes the TATA site; ‘exchange’ of H2A/H2B dimers with

H2A.Z/H2B dimers opens the DNA; ‘dissociation’ results in unbound DNA.


Transcriptional Activation from Poised Polymerases RNA polymerases are also bound to “transcriptionally silent” genes

that initiate transcription but do not transition to elongation. These polymerases are ready for transcription but are poised by

inhibitory factors. Gene transcription at the level of elongation may be important in

activation of genes.


Nucleosomal landscape of yeast genes. Top: RNA pol II transcribed

DNA region with nucleosome positions (gray ovals). Bottom:

Probability of histone binding (blue) with high levels of H2A.Z and histone acetylation, and H3K4

methylation in green.


Transcriptional Repression Histone

deacetylases (HDACs) remove acetyl groups and repress transcription. HDACs are subunits of

larger complexes acting as corepressor.

Corepressors are recruited to specific gene loci by transcription factors that cause the targeted gene to be silenced.

Transcriptional control Transcriptional repression

Transcriptional repression model.

Loss of acetyl groups and

addition of methyl groups leads to

chromatin inactivation and gene silencing.


DNA methylation Carried out by DNA methyl-transferases to

silence transcription in eukaryotic cells. Methylation patterns of gene regulatory

regions change during cellular differentiation. Activity of certain genes varies according to

changes in DNA methylation. DNA methylation serves more to maintain a

gene in an inactive state rather than to initially inactivate it.

DNA methylation is not an universal mechanism for inactivating eukaryotic genes.


Changes in DNA methylation levels during mammalian development


• Genomic Imprinting– Activity of certain genes, called

imprinted genes, depends on whether they originated with the sperm or egg.

– Active and inactive versions of imprinted genes differ in their methylation patterns.

– Disturbances in imprinting patterns have been implicated in a number of rare human genetic disorders.


Changes in DNA methylation levels during mammalian development

Long noncoding RNAs (lncRNAs) can be involved in genomic imprinting and X chromosome inactivation.

Most lncRNAs are associated with gene repression. lncRNA HOTAIR has a 5’ end that associates with PRC2 (histone methyltransferase for

H3K27) and 3’ end that binds CoREST (histone demethylase for H3K4). Global response: 700 genes in fibroblasts are bound by the PRC2-HOTAIR-CoREST

complex.


An lncRNA acting as a

mediator of transcriptional

repression


(6.5) Processing Control

Protein diversity can be generated by alternative splicing. Alternative splicing can become complex, allowing different

combinations of exons in the final mRNA product. There are factors that can influence splice site selection. Exonic splicing enhancers serve as binding sites for regulatory

proteins.


Alternative splicing. The Drosophila

Dscam gene illustrates the

diversity of transcripts (38,106) from a single gene

Processing ControlAlternative splicing

“Weak” splice sites can be used if the proper proteins are present.

Activation: SR proteins bind to exon and intron splicing enhancers (ESEs and ISEs) to stimulate splicing.

Repression: hnRNP proteins bind to exon and intron splicing silencers (ESSs and ISSs) to by-pass splicing.


Mechanisms of alternative

splicing. (Top) 5’ splice site

sequence changes can affect pairing

with U1 snRNA. (Bottom) Different

strength splice sites can be repressed or

activated depending on

proteins


RNA Editing Specific nucleotides can be converted to other nucleotides through mRNA editing. RNA editing can create new splice sites, generate stop codons, or lead to amino acid

substitutions. It is important in the nervous system, where messages need to have A converted to I

(inosine) to generate a glutamate receptor. Resultant glutamate receptor’s internal channel is impermeable to calcium ions. Conversion to apolipoprotein B mRNA to a truncated version in the intestines

(apolipoprotein B-48) produces a smaller protein that works to help absorb fats.

Processing Control RNA editing


The Control of mRNA Translation Several important processes depend on mRNAs that were synthesized

at a previous time and stored in the cytoplasm in an inactive state. Other mechanisms influence the rate of translation of specific mRNAs

through proteins that recognize specific elements in the UTRs of those mRNAs.

Example: mRNA that codes for ferritin.

(6.6) Translational control

A model for the mechanism of translational activation of mRNAs following fertilization of a Xenopus egg. mRNAs are maintained in the cytoplasm in an

inactive state by their short poly(A) tails and a bound inhibitory protein Maskin.


The Control of mRNA Translation When iron concentrations are low, iron

regulatory protein (IRP) binds to the iron-response element (IRE) to prevent translation.

When iron becomes available, it binds to the IRP, changing its conformation and causing it to dissociate from the IRE, allowing the translation of the mRNA to form ferritin.

Translational controlTranslation

The control of ferritin mRNA translation

Translational controlCytoplasmic localization


Regulated mRNA translation after transport from nucleus to the cytoplasm. Control occurs via interactions of specific mRNAs and proteins in the cytoplasm. Regulatory proteins act on unstranslated regions (UTRs) at both 5’ and 3’ ends. UTRs contain nucleotide sequences used to mediate translational-level control.

Cytoplasmic Localization of mRNAs In the fruit fly embryo the development of anterior-posterior axis is regulated by the

localization of specific mRNAs along the axis in the egg. Cytoplasmic localization of mRNAs is determined by their 3’ UTRs.

bicoid

oskar

Cytoplasmic localization of mRNAs. Schematic drawings showing three stages in the

life of a fruit fly: the egg, larva, and adult. The

segments of the thorax and abdomen are indicated.

Translational controlCytoplasmic localization


Regulated mRNA translation after transport from nucleus to the cytoplasm. Control occurs via interactions of specific mRNAs and proteins in the cytoplasm. Regulatory proteins act on unstranslated regions (UTRs) at both 5’ and 3’ ends. UTRs contain nucleotide sequences used to mediate translational-level control.

Cytoplasmic Localization of mRNAs In the fruit fly embryo the development of anterior-posterior axis is regulated by the

localization of specific mRNAs along the axis in the egg. Cytoplasmic localization of mRNAs is determined by their 3’ UTRs.

Localization of bicoid mRNA

(anterior pole) and oskar mRNA (posterior pole)

Localization of β–actin mRNA (red) near the

leading edge of a migrating fibroblast


The Control of mRNA Stability The lifetimes of eukaryotic mRNA vary widely.

Fos mRNA (cell cycle-related) is 10-30 minutes. Hemoglobin mRNA is greater than 24 hours.

Poly(A) tail length may influence the longevity of mRNA. As an mRNA remains in the cytoplasm, its poly(A) tail tends to be reduced. When the tail is about 30 A residues, the tail is shortened.

Certain destabilizing proteins in the 3’ UTR may affect the rate of poly(A) tail shortening. Globin mRNA 3’ UTR contains CCUCCU repeats that serve as binding sites for stabilizing

proteins. Short-lived mRNA 3’ UTRs have AU-rich regions that destabilize it. Introduction of a destabilizing sequence into the globin mRNA 3’ UTR shifts the half-life from 10

hours to 90 minutes.

Translational controlmRNA stability

• The Control of mRNA Stability– Deadenylation, decapping, and 5’ 3’ degradation occur

within small transient cytoplasmic granules (P-bodies).– P-bodies can also serve to store mRNAs for later translation.

Translational controlmRNA stability

mRNA degradation in mammalian cells: schematic series of events and fluorescent detection of P-

bodies with a GFP-DCP-1 marker (decapping protein)



The Role of MicroRNAs in Translational-level Control miRNAs act by binding to site in

the 3’UTR of their target mRNAs. Suppress gene expression by

either promoting deadenylation and degradation, inhibiting the initiation of translation, inhibiting elongation, or possibly activating degradation of nascent peptides.

Translational controlmicroRNAs

miRNA mediated gene silencing. miRNAs pair with sequence elements

within the 3’ UTR of target mRNAs

(6.7) Post-translational Control: Determining Protein Stability


The factors that control a protein’s lifetime are not well understood.

Protein stability may be determined by the amino acids on the N-terminus.

Degradation of proteins is carried out within hollow, cylindrical proteasomes.

Proteasome structure and function. High-resolution EM of an isolated Drosophila

proteasome (left) and model of a proteasome based on high-resolution electron microscopy

and X-ray crystallography (right)

Proteasome-mediated degradation: 1, protein is ubiquitinated; 2, protein binds to proteasome cap; 3, unfolded polypeptide enters proteasome; 4/5,

catalytic β subunits degrades protein

Post-translational Control: Determining Protein Stability


Proteasomes recognize proteins linked to ubiquitin.

Ubiquitin is transferred by ubiquitin ligases to proteins being degraded.

Once polyubiquitinated, a protein is recognized by the cap of the proteasome.

Once degraded, the component amino acids are released back into the cytosol.

Lys

Cap: removes chain

: protease

ATP depende

nt

Synopsis


In bacteria, genes are organized into regulatory units called operons. The nucleus of a eukaryotic cell is a complex structure bounded by the

nuclear envelope, which controls the exchange of materials between the nucleus and cytoplasm, maintaining the unique composition of the cell’s two major compartments.

The chromosomes of the nucleus contain a defined complex of DNA and histone proteins that form characteristic nucleoprotein filaments, representing the first step in packaging the genetic material.

Chromatin is not present in cells in the highly extended nucleosome filament but is compacted into higher levels of organization.

Mitotic chromosomes possess several clearly recognizable features. The nucleus is an ordered cellular compartment. The rate of synthesis of a particular polypeptide in eukaryotic cells is

determined by a complex series of regulatory events that operate primarily at three distinct levels.

Synopsis


All cells of a eukaryotic organism retain a full set of genetic information. Different genes are expressed by cells at different stages of development, by cells in different tissues, and by cells exposed to different stimuli.

Determination of the three-dimensional structure of a number of complexes between transcription factors and DNA indicate that these proteins bind to the DNA through a limited number of structural motifs.

The activation and repression of transcription is mediated by a number of large complexes that function as coactivators or corepressors.

Eukaryotic genes are silenced when the cytosine bases of certain nucleotides residing in GC-rich regions are methylated.

Alternative splicing in which a single gene can encode two or more related proteins allows relatively small genomes to encode a large diversity of proteins.

Gene expression is regulated at the translational level by various processes, including mRNA localization, control of translation of existing mRNAs, and mRNA longevity.

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