Trends in Biomedical Science

04 Epigenetics – Gene Expression and Regulation

Gene Expression and RegulationAdapted from: Hoopes, L. (2008) Introduction

to the gene expression and regulation topic room. Nature Education 1(1):160

http://www.nature.com/scitable/nated/article?action=showContentInPopup&contentPK=28455

The study of gene expression. • How does a gene know when it should express

itself? • How does this gene cause the production of a

string of amino acids called a protein? • How do different types of cells know which

types of proteins they must manufacture?

Genes can't control an organism on their own interact with and respond to the organism's

environment. Constitutive genes - always "on," regardless of

environmental conditions. o among the most important elements of a cell's genome, o control the ability of DNA to replicate, express itself,

and repair itself. o also control protein synthesis and much of an

organism's central metabolism. Regulated genes are needed only occasionally

cell-cell differences are determined by expression of different sets of genes. eg, an undifferentiated fertilized egg looks and acts quite

different from a skin cell, a neuron, or a muscle cell because of differences in the genes each cell expresses.

A cancer cell acts different from a normal cell: It expresses different genes. o microarray analysis, helps diagnosis and selection of

appropriate cancer treatment.• in eukaryotes, the default state of gene expression is

"off" rather than "on".

Histones - among the most evolutionarily conserved proteins known; Essential for the well-being of eukaryotes Have little change. A gene is tightly bound with histone, that gene is

"off." The histone code allows genes to be turned on. • includes modifications of the histones' positively

charged amino acids to create some domains in which DNA is more open and others in which it is very tightly bound up.

DNA methylation - appears to be coordinated with histone modifications, • lead to silencing of gene expression.

Small noncoding RNAs such as RNAi can also be involved to form "silent" chromatin.

Acetylation of the tails of histone molecules at specific locations, • less interaction with DNA,

thereby leaving it more open.

Complexes of proteins called chromatin remodeling complexes use ATP to repackage DNA in more open configurations.

It is possible for cells to maintain the same histone code and DNA methylation patterns through many cell divisions.

Epigenetic changes cause many human diseases

For transcription - the area around a prospective transcription zone needs to be unwound. complex process requiring the

coordination of ohistone modifications, o transcription factor binding and oother chromatin remodeling activities.

Specific proteins bind to specific DNA sequences if the DNA is open and accessible. Many of these proteins are activators, while others are

repressors; In eukaryotes, all such proteins are often called

transcription factors (TFs). Each TF has a specific DNA binding domain that recognizes a 6-

10 base-pair motif in the DNA, as well as an effector domain. Can find a footprint of a TF if that protein binds to its

matching motif in a piece of DNA. Also see whether TF binding slows the movement of DNA

in gel electrophoresis.

Activating TF, the effector domain recruits RNA polymerase II,

o begins transcription of the corresponding gene. Some activating TFs turn on multiple genes at

once. All TFs bind at the promoters just upstream of

eukaryotic genes. o they also bind at regions called enhancers,

oriented forward or backwards located upstream or downstream or in the introns of a

gene.

Many genes are coregulated, • Studying gene expression across the whole

genome via microarrays or massively parallel sequencing allows investigators to see which groups of genes are coregulated during differentiation, cancer, and other states and processes.

Small noncoding RNAs regulate gene expression – in most eukaryotes. Eg. the enzyme Dicer finds double-stranded

regions of RNA and cuts out short pieces that can serve in a regulatory role.

Argonaute is another enzyme that is important in regulation of small noncoding RNA–dependent systems.

Imprinting - another process involved in eukaryotic gene regulation; silencing of one of the two alleles of a gene for a cell's

entire life span. affects a minority of genes, but includes several important

growth regulators. For some genes, the maternal copy is always silenced,

while for different genes, the paternal copy is always silenced.

The epigenetic marks placed on these genes during egg or sperm formation are copied into each subsequent cell,

• affect these genes throughout the life of the organism

X inactivation causes some genes to be silenced for an organism's entire lifetime. Eg female mammals, one of the two copies of the X

chromosome is shut off and compacted greatly. process requires

o transcription, o the participation of two noncoding RNAs

one of which coats the inactive X chromosome

o a DNA-binding protein called CTCF.

• possible role of regulatory noncoding RNAs

• Chromatin Remodeling in EukaryotesAdapted from:

Phillips, T. & Shaw, K. (2008) Chromatin Remodeling in Eukaryotes. Nature Education 1(1):209

http://www.nature.com/scitable/topicpage/chromatin-remodeling-in-eukaryotes-1082

DNA strand in a single human cell is several meters in length.

coils many times to fit into the cell's nucleus the DNA winds itself around proteins called

histones, o complex known as chromatin.

chromatin • condense DNA within the nucleus, • control how that DNA is used.

– specific genes are not expressed unless they can be accessed by RNA polymerase and proteins known as transcription factors.

– tight coiling limits the access of these substances to eukaryotic DNA.

– a cell's chromatin must "open" in order for gene expression to take place.

– process of "opening" is called chromatin remodeling, – very important to the proper functioning of all eukaryotic cells. – different protein complexes, histone variants, and biochemical

modifications affect this process.

chromatin remodelers • are large, multiprotein complexes • use the energy of ATP hydrolysis to mobilize and

restructure nucleosomes. • nucleosomes wrap 146 base pairs of DNA in

approximately 1.7 turns around a histone-octamer disk,

• DNA inside each nucleosome is generally inaccessible to DNA-binding factors.

• Remodelers provide access to the underlying DNA to enable transcription, chromatin assembly, DNA repair, and other processes.

• remodelers convert the energy of ATP hydrolysis into mechanical force to mobilize the nucleosome,

• different remodeler complexes select which nucleosomes to move and restructure.

Remodelers - five families, specialized biological roles.

• all remodelers contain a subunit with a conserved ATPase domain

• each remodeler complex has unique proteins that specialize it for its unique biological role.

• ATPase domains of remodelers are similar in sequence and structure to known DNA-translocating proteins in viruses and bacteria.

remodeler ATPases are directional DNA translocases that are capable of the directional pumping of DNA. – binds approximately 40 base pairs inside the

nucleosome, – pumps DNA around the histone-octamer surface. – moves nucleosome along the DNA, – exposes the DNA to regulatory factors.

additional domains and proteins attached to the ATPase

• important for nucleosome selection, • help regulate ATPase activity. – bind to histones and nucleosomal DNA, – binding is affected by the histone modification

state. – helps determine whether the nucleosome is an

appropriate substrate for a remodeler complex

Nucleosomes, Histones, and Gene Transcription

a) Remodeling complexes of the SWR1 family can remove the canonical H2A–H2B dimers and replace them with Htz1–H2B dimers, with tails that might bind special regulatory proteins (Reg).

b) Nucleosome modification (only acetylation [Ac] is shown for simplicity) allows the binding of regulatory factors, which have specialized domains (bromodomains) that recognize acetylated histone tails.

c) Nucleosome repositioning allows the binding of a regulatory factor to its site on nucleosomal DNA. Remodelers are necessary to provide rapid access to nucleosomal DNA by sliding of the octamer along the DNA.

Chromatin structure highly complex changes quickly and easily. very important in directing some

elements of transcriptional specification.

An octamer of histone proteins usually forms the core of the nucleosomes

• established during DNA replication. • histones can be replaced by histone

variants or modified by specific enzymes, • making DNA more or less accessible to

the transcriptional machinery.

Histone Modification and the Histone Code

The N terminus.

a) General chromatin organization. Like other histone tails, the N terminus of H3 represents a highly conserved domain that is likely to be exposed or extend outwards from the chromatin fiber. A number of distinct post-translational modifications are known to occur at the N terminus f H3 including acetylation (flag), phosphorylation (circle) and methylation (hexagon). Other modifications are known and may also occur in the globular domain. b) The N terminus of human H3 is shown in single-letter amino-acid code. For comparison, the N termini of human CENP-A, a centromere-specific H3 variant, and human H4, the nucleosomal partner to H3, are shown. Note the regular spacing of acetylatable lysines (flag), and potential phosphorylation (circle) and methylation (hexagon) sites. The asterisk indicates the lysine residue in H3 that is known to be targeted for acetylation as well as for methylation; lysine 9 in CENP-A (bold) may also be chemically modified.

• histone variants have been found and localized to specific areas of chromatin. – eg, H2A.Z is a variant of H2A and is often enriched

near relatively inactive gene promoters. – eg, CENP-A is a histone variant • associated with centromeres. • localized through immunofluorescence studies, • has sequence homology to H3, • suggesting that CENP-A replaces H3 near the

centromere.

Histone sequences are highly conserved. Extending from each of the core histones is a

"tail," called the N-terminal tail. The C terminus forms a globular domain that

is packaged into the core. The N-terminal tail is more flexible and can

interact more with DNA and the different proteins within the nucleus.

Histone modification involves covalent bonding of various functional groups to the free nitrogens in the R-groups of lysines in the N-terminal tail. differing levels of acetylation and methylation on

the histones alter DNA transcription many more types of modifications have also been

observed, including phosphorylation, a common posttranslational modification.

The different types of modificationso called the "histone code,"o put in place and removed by a variety of different

enzymes

Nucleosome Positioning and Reorganization

Positive charges of the histones tightly bind the negative charges of the DNA, nucleosomes stop transcription factors that

need to bind to certain regions of DNA acetylations can remove the positive charge

on the lysine amino group that is acetylated, the nucleosome binds less to the DNA. This

modification results in a loosening of the coil.

Remodeling enzyme complexes slide the nucleosomes along the DNA to clear them from the promoter regions.

Chromatin remodeling activity by SWI/SNF or other remodeling machines can also be required for recruiting additional chromatin remodeling activity to the site.

Modifications at a promoter can occur in multiple steps that are independently regulated.

Changes in gene expression during the specific developmental stages of an organism or cell coincide with fluctuations in the levels of each of the specific protein complexes involved in chromatin remodeling.

Signaling Function of Remodeled Chromatin

• Histone modification can open chromatin, thus permitting selective binding of transcription factors that, in turn, recruit RNA polymerase II.

• Varying levels and types of histone modifications have been shown to correlate with levels of chromatin activation.

Type of Histone

modification H3K4 H3K9 H3K14 H3K27 H3K79 H3K36 H4K20 H2BK5

mono-methylation activation activation   activation activation   activation activation

di-methylation   repression   repression activation      

tri-methylation activation repression   repression

activation

activation   repression

repression

acetylation   activation activation activation        

DNA Methylation

• Transcription is also controlled through methylation of the DNA strand itself.

• This involves cytosine bases of eukaryotic DNA being converted to 5-methylcytosine.– gives repression of transcription, particularly in

vertebrates and plants. • The altered cytosine residues are usually next to

a guanine nucleotide, – This gives two methylated cytosine residues set

diagonally to one another on opposing DNA strands. – CpG groups

Heavily methylated regions of DNA with elevated concentrations of CpG groups are often found near transcription start sites.

proteins that bind to methylated DNA also form complexes with proteins involved in deacetylation of histones. o so, when the DNA is in a methylated state, nearby

histones are deacetylated, resulting in compact, semipermanently silent chromatin.

o or, demethylated DNA does not draw deacetylating enzymes to the histones, but it often attracts histone acetyltransferases, allowing histones to remain acetylated and promoting transcription.

Epigenomics can help show

how neurons grow in a cell culture from stem cells - cells that have the capacity to mature into any cell type.

the epigenomic signatures that give rise to the first major split in cell lineage within stem cells during embryonic development o ectoderm, endoderm and mesoderm - the cells that will go

on to form the nervous system, organs, and muscles respectively

o helps find the "recipe" to mix the correct genes to make different cell types.

genetic variation in the DNA-changes in the As, Gs, Ts, and Cs-that interacts with epigenetic chemicals and how those changes could be linked to autoimmune disease.

epigenomic changes in cancer cells. o can predict the kinds of mutations of a particular cancer

based on the epigenome of the type of cell from which the cancer most likely originated.

o the epigenome of the cancer's ancestral cell has a large influence on which genes within a tumor mutate over time different from cancer-cell lines, the standardized cultured cells

grown in laboratories, which are often studied as models for cancer. important implications in cancer that may not be applicable for

study in laboratory-adapted cancer-cell lines.

• epigenetic changes in the brain during Alzheimer's neurodegenerationo the altered epigenome in the mouse brain was

similar to samples from dead Alzheimer's patients.

o epigenetic pattern different in tissue from adults that did not suffer from Alzheimer's disease.

o this may lead scientists to new targets for drug discovery to combat Alzheimer's.

Summary • Storage of eukaryotic DNA in small, compact

nuclei requires that this DNA be tightly coiled and compacted in the form of chromatin. However, the structure of chromatin also appears to serve a second, possibly more important role, in that it gives eukaryotic cells complex levels of control over gene expression.

Chromatin and the DNA sequences it contains are constantly undergoing modifications, thereby periodically exposing different regions of DNA to transcription factors and RNA polymerases.

The cumulative effects of these changes are various states of transcriptional control and the ability of eukaryotic cells to turn genes on and off as needed. This complexity provides eukaryotes with a means of making the most of a relatively small number of genes.

However, much research remains to be performed before investigators precisely understand how the many mechanisms of chromatin remodeling operate, as well as how they work together to result in the complex patterns of gene expression characteristic of eukaryotic cells.