Chromosomes and Gene Regulation

All cells can use their genes selectively – turning some on and keeping others off.

In multicellular organisms, gene expression is under complex controls. All cells have the same DNA sequences, they same chromosomes, and yet they each look and function very differently.

Cell differentiation is achieved by changes in gene expression. The differences between this neuron and the lymphocyte depend on the precise control of gene expression.

Same genome

Different genes expressed

Questions 2-4,6,7-9,11,12,15

Eucaryotic DNA is packaged into Chromosomes. Human cells contain two of each chromosome, one maternal and one paternal – homologous chromosomes Sex chromosomes are non-homologous chromosomes, X from mom, Y from dad.

Bacteria typically have one circular DNA molecule. It is also associated with proteins that condense the DNA but less is know about the structure.

Chromosomes are typically stained by dyes that distinguish between areas rich in A-T nucleotide

pairs and areas rich in C-G pairs. This results in a pattern of banding that is unique to each chromosome. Cytogeneticists use these to detect major chromosomal abnormalities.

centromere

large rRNA

Cells can vary the structure of their chromosomes for DNA replication and for gene regulation. Chromatin = DNA plus proteins, the stuff chromosomes are made of.

The state of condensation of chromosomes varies according to the cell growth cycle. The mitotic chromosomes are highly condensed, in contrast to that of the interphase chromosome.

The condensed state is important, allowing the duplicated chromosomes to be separated

Three types of specialized sequences found in all eucaryotic chromosomes ensure that chromosomes replicate efficiently.

many, to ensure speed

kinetochore = protein complex that binds the spindle and the centromere

DNA polymerase requires a primer at the end of the chromatin. Without telemers, chromosomes would continually shorten.

Also protects ends from attack by the DNA-digesting enzymes – nucleases.

A. chromatin spilling out of a lysed interphase nucleus

B. a mitotic chromosome, which is duplicated already

A. chromatin isolated from an interphase nucleus = 30 nm

B. chromatin “unpacked” after isolation

The nucleosome is the first and most fundamental packing level of chromatin.

Histones are small proteins with a high proportion of positively charged amino acids (lysine and arginine). These genes are the most highly conserved of all know eucaryotic proteins.

Form a histone octamer

1/3 the lengthChromosomes have several levels of DNA packing. Histons H1 pulls the other histones together to form the typical 30-nm chromatin fiber

In the more compact state, RNA polymerase etc can not bind, and transcription stops.

cytoskeleton organizes the chromatin

A typical mitotic chromosome

• Interphase chromosomes contain both condensed and more extended forms of chromatin– highly condensed interphase chromatin, is called

heterochromatin and makes up about 10% of the chromatin and is transcriptionally inactive.

• normally active genes moved to this region are inactivated– euchromatin = all of the chromatin except

heterochromatin, varies– regions that are actively being transcribed into RNA or

easily available for transcription are more extended (10%) – active chromatin [30-nm fiber or 300nm]

• transcription (RNA polymerase) and replication (DNA polymerase are not obstructed by histones. It is thought that the DNA partially detaches from the histone core as a polymerase moves through, and reassembles immediately.

Happens early in development

Female becomes a mosaic, half of the cells in each organ are maternal, half are paternal

Position effects

A. ADE2 encodes an enzyme whose absence leads to the accumulation of a red pigment.

B. The white gene controls eye pigment production. When inactivated the flies have white eyes.

It is thought the heterochromatin areas can spread and control the expression of genes near them

Interphase chromosomes are organized within the nucleus. The nuclear envelope is supported by two network of protein filaments, the nuclear lamina inside and intermediate filaments outside the membrane. Nuclear pores actively and specifically transport.

The interior is not a random jumble of DNA, RNA, etc. It is thought that DNA is organized by attachment of parts of the chromosomes to sites on the nuclear envelope or the nuclear lamina.

Gene regulation:

As an organism develops, cells differentiate into various types of cells.

Differentiation arises because cells make and accumulate different sets of RNA and protein molecules – they express different genes.

Most of the cells in an organism contain the entire genome. The difference between a muscle cell and a neuron depends on gene regulation.

Gene (eucaryotic) control.

Transcription is controlled by proteins binding to regulatory DNA sequences.

Promotor includes

RNA polymerase binding site

initiation site

Regulatory DNA sequences bound by gene regulatory protiens

some short – simple gene switches

some long and complex (eucaryotic)

molecular microprocessors which respond to a variety of signals, integrate them, and determine the rate of transcription.

Expression depends on

cell type

its environment

its age

extracellular signals

Edge of bases

Gene regulatory proteins insert into the major groove, contacts and binds (noncovalently) to the edges of the bases (about 20 interaction), usually without disrupting the hydrogen bonds. This binding is very strong and very specific for the nucleotide sequence. Frequently DNA-binding proteins contain alpha-helices bind in pairs - dimerization - which doubles the contact area, increasing the strength and specificity of the interaction.

Homeodomain – a structural motif

Zinc finger motif

Leucine zipper motif

An operon = a cluster of bacterial or viral genes transcribed from a single promoter.

A sequence of DNA within the promotor is recognized by regulatory proteins - the operator.

Repressor protein switch genes on or off.

Is recognized well by RNA polymerase.

Bacteria in your gut after you eat a steak

Regulatory proteins like these are allosteric.

These regulators allow fast response to the environment because they are always present in the cell - constitutively expressed

Activator proteins act on promoters that do not bind RNA polymerase on their own. These promoters are made fully functional by the addition of a bound activator protein which is also allosteric, activated by binding another molecule at a site different from the DNA binding site.

Example: CAP has to bind cyclic AMP (an intracellular signaling molecule) to bind DNA. Genes regulated by CAP are switched on in response to increases in cAMP, which is triggered by signals received by cell membrane receptors.

Initiation of transcription in eucaryotic cells is more complex as compared to the simple, economical procaryotic genetic switches.

1. Three RNA polymerases (Table 1)

2. Eucaryotic RNA polymerases require general transcription factors to bind the promoter.

3. Gene regulatory proteins (repressors and activators) can influence initiation of transcription from regulatory sequences found thousands of nucleotide pairs away from the promoter - enhancers.

4. Transcription is impacted by the chromatin structure.

General transcription factors assemble on all promoters transcribed by RNA polymerase II

Start with binding of TFIID which binds a short DNA sequence with many A-Ts - TATA box - typically 25 nucleotides upstream from the transcription start site. This binding causes a dramatic distortion in the DNA. Figure 24.

Other factors assemble along with RNA polymerase = transcription initiation complex. TFIIH contains a protein kinase subunit which adds phosphates to RNA polymerase, releasing it from the complex to start transcription

Start with binding of TFIID which binds a short DNA sequence with many A-Ts - TATA box

This binding causes a dramatic distortion in the DNA.

TBP is a subunit of TFIID which binds the TATA box.

Partially unwound areas

A model for gene activation from an enhancer

General transcription factors usually can not efficiently initiate transcription alone.

Enhancers can be thousands of base pairs away, either upstream or downstream and can either increase or decrease transcription.

“Action at a distance”

Chromatin structure effects gene transcription.

The presence of nucleosomes does not generally block elongation but they may inhibit initiation if they are positioned over a promoter

The effect of chromatin structure on transcription initiation is not well understood yet. More compact chromatin does block transcription.

The regulatory mechanisms involved in packaging interphase chromatin (heterochromatin, inactive X) are also still a mystery.

Summary of gene activation and regulation in procaryotes and eucaryotes.

Gene Regulatory sequences of a typical eucaryotic cell

Regulated by combinations of proteins over up to 50,000 nucleotide pairs

Combinatorial control can be positive or negative

Model for the control of the human beta-globin gene

Some gene regulators like CP1 are present in many types of cells

Others like GATA-1 are only found in a few types of cells.

These are thought to contribute to the cell-type specificity of gene expression

The expression of different genes can be coordinated by a single protein in eucaryotic cells. This can result in rapid switching on or

off of whole groups of genes.

Glucocorticoid steroid hormone is released in response to starvation or intensive physical activity. This hormone stimulates many different cells to do many different things, including liver cells to increase expression of many different genes, some to stimulate the production of glucose from amino acids. All of these genes responding to glucocorticoid steroid are regulated by the binding of the hormone-glucocorticoid receptor complex to a regulatory site.

Regulatory proteins specific for this gene

Regulatory proteins (combination control) specific for this gene

Combinatorial control

many control one

Single protein control

one controls many

This system allows the dramatic differences between cell types to be controlled during differentiation and produced by differences in gene expression

Myoblasts (mucle precursor cells) fuse and produce actin and myosin, ion channel proteins, etc. One regulatory protein involved is MyoD.

Fiberblasts from the skin, transfected with MyoD behave like myoblasts. Other cells do not.

Stained green with an antibody that binds a muscle cell-specific protein.

Combinatorial gene control generates different cell types.

A limited set of gene regulatory proteins can control the expression of a much larger number of genes.

Different combinations will produce different phenotypes

Notice that each daughter cell remembers the previous gene is activated.

When differentiate cells divide, they produce the same type of cells. Muscle and Neurons do not divide, but others like fibroblasts, smooth muscle cells hepatocytes do. Two possible mechanisms include positive feedback loops and propagation of chromatin structure.

Positive feedback loops include proteins that regulate their own genes.

Model for propagation of a condensed chromatin structure. As in X chromosome inactivation.

The formation of an entire organ can be triggered by a single gene regulatory protein. Example, eye development in Drosophila, mice, and humans.