Bio 127 - Section IIntroduction to Developmental Biology

Developmental GeneticsGilbert 9e – Chapter 2

This chapter is all about making different cells from equivalent DNA sequence....

1. Every somatic cell has the complete genome

2. A small percentage of the genome is expressed by an individual cell type and a portion of what is expressed is unique to that cell type

3. Cell differentiation during development is the process by which cells select the DNA that makes them unique

4. Unused genes are not lost and the cell retains the potential to use them in the future

All non-sex cells have the same DNA – all of it – and are capable of reusing the parts they don’t use daily!

Genomic Equivalency of allsomatic cells was demonstratedby the cloning of Dolly fromadult mammary epithelium.

“somatic nuclear transfer”

orreproductive

cloning

Figure 2.2 The kitten “CC”

sheepcats

g. pigsrabbits

ratsmicedogs

horsescows

Differential Gene ExpressionA small percentage of the genome is expressed by any given cell and a portion of what is expressed is unique to that cell

So, where are the points of differential control?

1. Differential gene transcription to RNA

2. Selective RNA processing in the nucleus

3. Selective mRNA translation in the cytosol

4. Differential peptide modifications

The steps in gene expression

1

2

3

4

Unlike the prokaryotes,our DNA is complexed50:50 with protein in avery highly regulatedstructure called chromatin

Control of transcriptionby histone modification

Heterochromatin is too tight packed to transcribe easily

Euchromatin is accessible

3 Stages of Transcription 1. Initiation 2. Elongation 3. Termination

Acetylation promotes Initiation Methylation can go either way

(lysine amino acid residues)

People are figuring out the “histone code”

Differential Gene Transcription: Epigenetic Memory

OK. So a cell differentiates to become a blood vessel smooth muscle cell......

• How come all of its mitotic descendents don’t have to go through differentiation?

• Trithorax proteins bind to open nucleosomes and keep them open.

• Polycomb proteins methylate nucleosomes and then bind to them to keep them tight.

• These effects can then be directly passed through mitotic cell division to the offspring.

What mRNA sequence makes up the start codon? the stop codon?

What amino acid do they produce?

Anatomy of a Gene

Control of Gene Transcription at the Promoter

Nucleotide Sequence Nomenclature

Exon meanssequence thatexits the nucleus

Intron meanssequence thatstays insidethe nucleus

Standard FormattingThis shows the ‘sense’strand, the DNA thatmatches the RNA. It is NOT the DNA strandthat is used to templatethe RNA!

Differential Gene Transcription: Gilbert Terminology

• Promoter– Binding sites for TF II family transcription factors– Site of RNA Pol II recruitment, stabilization, activation– Made up TATAbox and CpG islands

• Enhancer– True transcriptional determinant– Binding sites for tissue-specific transcription factors– Recruit histone acetyltransferases to unwind DNA– Stabilize Transcription Initiation Complex

Differential Gene Transcription: The eukaryotic transcription pre-initiation complex

The TranscriptionInitiation Complexforms on every genethat gets expressed.Its presence there isreally determined bythe tissue specifictranscription factorsthat bind to enhancercis-elements.

The TF II proteinsare commonly calledGeneral TranscriptionFactors.

TF-II proteins and RNA polymerase can only bind promoters positively identified by tissue specific transcription factors

Tissue-Specific Transcription Factors

How do Transcription Factors Function?

1.The primary feature is DNA binding domain sequence homology.

- Small changes in sequence in this domain can significantly alter DNA binding site sequence.

2.The trans-acting domain.- Recruits acetyl- and/or methyl-transferases to loosen nucleosome

- Stabilizes the TF-II pre-initiation complex

3. The protein-protein interaction domain- Dimerization, combinatorial functions, rate control

Three-dimensional model of the homodimeric transcription factor MITF (one protein in red, the other in blue) binding to a

promoter element in DNA (white)

Differential Gene Transcription: Enhancer Modularity

Enhancer sequence is the same in all cells

A. Most enhancers have many tissue-specific TF binding sites– The combination of tissue-specific TFs present determines

the rate of transcription in that cell type• If A,B,C present than high transcription rate• If A,B,Z present than low transcription rate• If A,Y, Z present than zero transcription rate

B. Some genes also have multiple enhancers– The combination of tissue-TFs present determines the

presence of transcription in different cell types• If A,B,C present than high transcription rate• If X,Y,Z present than zero transcription rate

Example A. Modular Enhancers

Pax-6, Sox2 and L-Maf are all required for crystallin expression

Pax-6, Pbx1 and Pdx1 are all required for somatostatin expression

Example B. Multiple Enhancers in a Gene

The pax-6 gene hasfour enhancers and isexpressed exclusivelyin those four tissue types.

Pax-6 has a positive feedback loop

A really important idea in cell differentiation is that there must be a molecular mechanism that keeps a cell differentiated.

– The pax-6 gene has a Pax-6 site in its enhancer

– When it is present the transcription rate is maximal

– This mechanism is repeated in several cell types

Differential Gene Transcription: Coordinated Expression

• Put what you know about cells, enhancers and TFs together....

– What do you think the enhancers of genes for SkM actin and SkM myosin are like?

– How about for SkM actin and SmM actin?

How Does a Cell Change Its Transcription: Silencers

Zinc-fingerNRSF bindsto the NRSEand stopstranscription

“Pioneer” Transcription Factors

MyoD and E12displace the inhibitor as longas Pbx gets therefirst.

DNA methylation can block transcription by preventing transcription factors from binding to the enhancer region

DNA Methylation of Globin Genes in Development

Transmitting DNA methylation to daughter cells

Modifying nucleosomes through methylated DNA

DNA methylationcan lead indirectly tohistone methylationthrough recruitmentof transferase activity.

Differential Gene Transcription: X-inactivation and Genomic Imprinting

• X-chromosome inactivation– Promoter methylation in one X-chromosome– Random between maternal and paternal– Achieves X-linked Dosage Compensation

• Genomic Imprinting– Re-distribution of methylation during gametogenesis – At least 80 genes in mammals– Only mom’s or only dad’s allele is expressed

Differential RNA Processing

Two major ways differential RNA processing can effect development

a. Selection and release of different sets of nRNA to the cytosol

b. Splicing different mRNAs from the same nRNA using different exons

Differential RNA Processing

Selection and release of different sets of nRNA to the cytosol

– More genes are transcribed in the nucleus than than are allowed to be mRNA in the cytosol

– The unused nRNAs are degraded or used for non-coding RNA species in the nucleus

Cell-Specific RNA Processing

Splicing different mRNAs from the same nRNA using different exons

– Alternative splicing occurs in ~92% of human genes

– “Splice sites” are formed from consensus sequences found at the 5’ and 3’ ends of introns

– Different splicosome proteins made in different cells recognize different consensus sequences

– The result is families of related proteins from the same gene in different cell types

Figure 2.26 Some examples of alternative RNA splicing (Part 1)

Figure 2.26 Some examples of alternative RNA splicing (Part 2)

Figure 2.27 Alternative RNA splicing to form a family of rat α-tropomyosin proteins

The Dscam gene of Drosophila can produce 38,016 different proteins by alternative nRNA splicing

The proteome in most eukaryotes dwarfs the genome in complexity!

Dscam protein is specifically required to keep dendrites from the same neuron from adhering to each other

Dscam complexityis essential to the establishment of the neural net by excludingself-synapses fromforming

Splicing Enhancers and Recognition Factors

- These work much like transcription enhancers and factors

- Enhancers are RNA sequences that bind protein factors to promote or silence spliceosome activity at splice site

- Many of these RNA sequences and trans-factors are cell type-specific, eg. muscle cells have specific sequences around all of their splice sites, thus make muscle-specific variants

Muscle hypertrophy through mispliced RNA

Splice sitemutationscan be verydeleterious,rarely can beadvantageous

Control of Gene Expression at the Level of Translation

A. Differential mRNA longevity can determine total protein expression

B. Selective inhibition of mRNA translation can determine time of expression

C. Selective destruction of active mRNA can determine cessation of expression

D. Selective placement of mRNA can determine localization of expression

E. Selective alteration of peptides post-translationally can determine cell-specific expression

Differential mRNA Longevity

- The longer the poly-A tail, the longer mRNA life

- Sequence of 3’-UTR determines length of tail

- External regulation: hormones, growth factors

- Internal regulation: stabilizing proteins

Figure 2.31 Degradation of casein mRNA in the presence and absence of prolactin

Selective Inhibition of mRNA Translation

- Dormant mRNA in oocyte awaits fertilization

- Proteins that control the cleavage cell cycle

- Proteins to control cell differentiation

- Ion signals that follow sperm entry set them off

Translational regulation in oocytes

Protein binding in Drosophila oocytes

Bicoid in flies acts like maskin - but only on the caudal gene!

Selective destruction if active mRNA: MicroRNAs

- Naturally occurring antisense RNA that stop expression of the mRNA they hybridize with

- Built-in system to actively shut down expression that you wanted earlier

- Can be found as different gene, in introns of same gene, even out in the “junk DNA”

- miRNA are transcribed, processed in nucleus, transported into cytosol and further processed there

Current model for the formation and use of

microRNAs

Figure 2.34 Hypothetical model of the

regulation of lin-14 mRNA

translation by lin-4 RNAs

miRNA complex can block translation in several ways

Identified Pathways which use miRNAs

- Larval development in C. elegans

- Cell fate decision in B and T lymphocytes

- Removal of maternal mRNAs after its needed

- Ventricle differentiation in the heart development

- Muscle cell division rate in response to myostatin

- Can even affect the methylation pattern in histones which can alter the entire gene expression pattern

The lymphoid precursor can generate B or T cells

Cytoplasmic Localization

- Most mRNAs are placed in specific locations in the cell prior to being translated

- 3 major mechanisms of localization1. diffusion and local anchoring through local protein trapping2. localized protection – degraded everywhere but target3. active transport along the cytoskeleton (the big one)

- Often secured once there in actin cytoskeleton

- Critical in brain, in the neuronal dendrite

Figure 2.38 Localization of mRNAs

Example: Stored mRNAs in Brain Cells

- mRNA actively transported to dendrite on microtubules

- Proteins that are critical in initial building of the synaptic connections

- The same process must be active for us to learn as the connections must be remade

-

Figure 2.39 A brain-specific RNA in a cultured mammalian neuron

Post-Translational Regulation of Gene Expression

• Let’s remember some basics....

- primary protein structure

- secondary protein structure

- tertiary protein structure

- quaternary protein structure