GCC550 Exam 4 Part III

The cell cycle

Features: cellular growth, DNA replication, chromosome segregation, an division of cell and contents

• single cell accumulating mass

• half of DNA segregation

• all components need to be segregated

Highly ordered series of events, highly conserved process

• needs to be unregulated cell division process

Necessary for Development replacement of adult tissue that has high cell turnover

• need to be able to make copies, organ system needs to be able to be replaced

Cell Cycle Phases

G1 - Gap phase 1, growth of cell

S - DNA synthesis

G2 - Gap phase 2, growth and preparation for mitosis

M - mitosis, cell division

• Each step has different checkpoints, and or cues

• problems in regulators leads to cancer

Mitosis vs Interphase

Interphase:

• G1: Cell grows and carriers out normal metabolism; organelles duplicate - is also carrying out cue, sensing environment for prep to divide 

• S: DNA replication and chromosome duplication - once DNA fully replicated

• G2: Cells grows and prepares for mitosis - replicated organelles, sense DNA synthesis, replication/ quality of replication.

Mitosis: Small steps for cell separation - Prophase, prometaphase, metaphase, anaphase, telophase, cytokinesis

Mitosis and Meiosis

M phase - 5% of the total cell cycle extensively studied because of mitotic chromosomes and their behavior

G0

Reversible cell cycle exit (post-mitotic cells)

• has all characteristics of G1

• cells reversibly enter

• neurons are typically in this state

G0 Cell cycle length vary dramatically

Non-dividing cells - nerve cells, muscle cells, RBCs. Cell that can be induced to divide-liver cells, B and T cells.

• Rapidly dividing cells - hematopoietic stem cells, epithelial cells and embryonic cells

• G0 time varies in cells

• Cell cycle length of typical mammalian cell cycle (S through Mitosis) 18-24 hours

How can we determine which stage of the cell cycle a cell is in?

DNA content:

• G → S → G2 → M

• 2N-2N-4N-4N—4N-2N

  • In s phase, DNA not fully duplicated 

• N - varies by species, but is equal to 1 haploid genome

Decisions, Checkpoints, and Transitions

G1 Checkpoints:

• Is environment good?

• Growth factors present?

• DNA damage?

S checkpoints:

• DNA damage?

• DNA replicated?

G2 Checkpoint:

• DNA Damage?

• DNA replication errors?

• Mitotic spindle properly formed?

M checkpoint:

• DNA replication errors?

• Mitotic spindle properly formed?

Checkpoints are enforced by regulation of CDK activities

Biochemical events in Cell Cycle Regulation

Proteins involved in checkpoints and regulating phases

• Kinases: cyclin-dependent kinases (cdks) 

  • cell cycle specific

  • specific cyclins for each cycle

  • specific ones for each cycle

  • Active kinases phosphorylates substrates, which in turn allows the cell to go through different stages of cell cycle

• There are multiple cdks for each cell cycle transition - multiple substrates known

• Cyclins bind CDK to activate

Active enzyme

A complex of cdk and cyclin

• set of machines made to regulate cell cycle

  • CDKs - always expressed

  • cyclins - phase dependent expression

• distinct from enzymes that replicate cell components a dedicated cell cycle control machine

• Regulatory mechanisms: expression, activity, interaction, degradation

Specific cell cycle transitions

Controlled by specific cyclin/cdk molecules

• G1: Cyclin D with CDK4/CDK6 - expressed late in G1

• G1/S: Cyclin E with CDK2

  • swaps cyclin partners

• S: Cyclin A with CDK2

• M: Cyclin B/A with CDK1

Regulation of Cyclin proteins

Cyclin levels can be regulated to determine if CDKs are active or not

• several ways to regulate:

  • Cyclin protein levels

  • Phosphorylation of cdks themselves

  • Inhibitory molecules

Cyclin protein accumulation

Regulates Cyclins

• Affects of cyclin mRNA transcription

  • cyclin mRNA translation

  • Other steps

• CDKs are always present but cyclins switch

Cyclin D1 transcription

Linked to many growth factor signaling pathways

• c-JUN, c-FOS, c-MYC

  • closely connected to signaling pathways

Cyclin protein degradation

Protein stability is regulated, multiple protein structures

• Cyclin is ubiquityated by APC/C

• Forms polyubiquitin chain on cyclin

  • spend energy to get rid of cyclin

  • gets degrated by proteasome

Cdk phosphorylation

Cdk is an enzyme can itself be post-translationally modified by phosphorylation

• there are 2 types of phosphorylation events inhibitory [Tyr 15] and stimulatory [Thr 161]

• de-phosphorylation of tyrosine 15 is able to phosphorylation important components for M phase

  • Inhibitory phosphorylation “wins” - active kinase must have stimulatory phosphorylation and NOT have inhibitory phosphorylation

  • Results in conformational change

Multiple steps in enzyme activation

A) Inactive (no cyclin bond)

B) Cyclin binds, Partly active - forms T loop (no longer blocking active site)

C) T-loop is phosphorylates - fully active form

Confirmational changes in cdk not cyclin

Inhibitory Molecules that Blocks Cdks

These proteins interact with cdk molecules and inhibit their activity

• Ex. p21, p27, p16 (CDKN1A, 1B…)

• Currently there are 16 known inhibitors

• Each cdk has its own group of inhibitory proteins

Inhibitors bind CDk/cyclin complex

Alter shape of pocket as a result

• ex: p27 Kip1 - cdk inhibitor forms c shape across proteins - blocks substrate

Inhibitors have structure based specificity

p16 INK4A binds specifically to CDK6

Inhibitors are tightly regulated

Active SCF complex can’t bind Cdk inhibitor protein for degradation unless there is a specific phosphorylation event

Focus on the G1 to S transition

Early in G1 - CycD/cdk4 becomes activated:

• phosphorylate substrates

• substrate phosphorylation permits cell cycle progression

One of the Substrates is Rb protein

pRb inhibit E2F - activation leads to pRb phosphorylation and dissociation from E2F

• outcome is turning on sets of genes that allow cell cycle progression

  • Cyclin E

  • DNA polymerase

  • thymidine kinase

• E2F drives transcription of genes in S phase

  • nucleotide biosynthesis

Regulation of G1/S phase transition by the RB protein

When hyper-phosphorylated, Rb is inactivated

• at R point

• multiple sites of phosphorylation

Alfred Knudson (1971) the two-hit hypothesis

Of tumor suppressors

3 Major regulatory transitions (checkpoints)

1) Start (G1 to S)

2) G2 to M

3) Metaphase Anaphase

p53-dependent checkpoint after DNA damage

Transcription factor, drivers cyclin inhibitor transcription, and complexes and inhibits CDK-cyclin complex

Cdk/cyclin complexes regulate RNA Pol II-based transcription

Cell cycle regulators influence DNA damage repair

Cell cycle regulators and epigenetic regulation

modify histones

Cell cycle regulators controlling stem cell self-renewal

stem cell function

Why choose a mouse as a model organism?

Similar to humans, have placenta, small, short generation time

• has human genetic and physiologic similarities

• are well documented, established tools, deep literature records

Genetic Modifications

Progression of available tools: Find a mutant or make a mutant

• Either look for a visible different gene

• Or use radiation, chemical, or transposon insertion to damage DNA and result in repair → cause changes in DNA sequence, required for a lot of screening

Genetic Modifications - Target a mutant

• Knock-out: use homologous recombination to introduce a disruptive sequence into a locus - target specific genes - make a null allele

• Knock-in: insert foreign DNA into a locus of choice

  • distinct from transgenesis where the insertion point is random, and does not disrupt a targeted locus

• Conditional Mutagenesis: selective knockout in a tissue, cell population, and/or developmental stage of choice

  • compare tissue-specific vs whole organism - YYI

Whole organism mutagenesis

YYI (and other gene) knock-out mice die near implantation

• YYI is an essential gene

• mice with mutant alleles where pari implantation lethal

Conditional mutagenesis

Cell population and or developmental stage restriction allow other tissues to function

• tissues to function

Embryonic Stem (ES) manipulation 

Requires drug selection, chimeric embryos, and back crossing

• embryos develop in uetero

• form chimeric mice

• cross with wildtype to obtain stain with expression in all cells

ES cells can be manipulated in vitro

Selected through drug resistance

• explain ES manipulation and husbandry to produce targeted KP mice

• explain role of G418 an FIAU selection to select for correctly targeted mutation

The gene targeting vector has 2 selection markers and homology to the target locus

Results in outside genes getting lost

• Targeting vector is inserted into target locus, disrupting the locus

Gene targeting vector has 2 selection markers and homology to target locus

Randomly integrated

Compare the incorporation of cassettes and predict consequences of selection

low efficiency

Predict outcome of genotyping

G418s has only 750bp

G418r has 1200bp (recombinant locus) - modified population

• has a mix of to fragments

Embryos bearing modified ES cells are cultivated in vivo

Pups requires back-crossing - genotyping mice

Match genotyping PCR outcome to mouse strains - 2

has amplification at 1200bp and 750bp

Match genotyping PCR outcome to mouse strains - 1

has amplification at 750bp

Determine the genotypes of these targeted mice

Using genotyping PCR to distinguish targeted and untargeted mice

• assign genotypes from sizes of PCR products

• Wildtype (+), recombinant (-)

  • heterozygous (has 2 bands)

  • WT homozygous' (has one band in between 800/600bp)

  • recombinant (has on band at 1300bp)

  • Recombinant

How does targeting vector disrupt a genome locus?

homology arms flank neo gene can be separately recombined with the genome locus through homologous recombination (HR, crossing over in meiosis)

• leads to neo incorporation of neo into genomic locus

• excised region is chosen to remove critical structural aspects of genomic locus gene product

Why does HR lead to disruption of the target locus, but integration to other genomic sites occurs through distinct mechanisms?

HR requires a significant amount of homology between the exchanging DNA molecules

• other genomic loci are not predicted to have significant homology with the vector

• integration of the targeting vector at these sits cannot use the HR mechanism

Mutagenesis using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

CRISPR uses guide RNA and PAM to recognize target sites - Cas9 performs enzymatic activity

• guide RNA stitched together - directs where we want Cas9 to go

• pairing of protospacer DNA with the guide RNA leads to activation of Cas9

  • PAM (protospacer adjacent motif) and complementary region lead to activation of Cas9 endonuclease activites

Repair Pathways introduce changes at the target genomic loci

Works for knock-in strategies, can go down two different pathways:

• NHEJ: stitch together - results in mutagenesis

• HDR: move access to repair template to stear towards it

Homology-directed repair using a co-delivered template

Used to make precise alterations

• introduces HM1 cut

• get high modification of fibroblast

Off-target Effects are a Danger

CRISPR is great - except genome is full of a lot of repeats

• 5bp closet to PAM must match exactly but mismatches closer to the 5’ end of the sgRNA are tolerated

• Chromatin accessibility (less efficient when sequences within condensed chromatin are targeted)

• lays chucks of repeats → predicts off target sites

“Nickase” Cas 9 mutations

inactivate a nuclease site and only cut one strand

• eliminates where cas9 cleaves

  • cleaves only target strand

• Individual point mutation in Cas9 lead to loss of nuclease activity and prevents double strand bearks

Staggered, single-stranded nicks

Nickase Cas 9 mutant, 2 sgRNAs and D10A

• restains off target effects

• off target is fixable by DNA ligase

CRISPR modification of pro-nuclear embryos

Can be used for germ-line incorporation

• these modifications can be adapted for mouse embryo genome engineering

How does guide RNA direct Cas9 endonuclease to genomic targets

Complementary base pairing between the RNA and the genome locus to high selectively for targeting 

• Cas9 is ineffective in the absence of these interactions

How does Cas9 activity lead to mutations?

Double stranded break introduced by Cas9 will recruit either non-homologous end joining (NHEJ) or homology-directed repair (HDR)

• NHEJ is usually mutagenic since bases are removed in the repair

• HDR can be supplemented with a repair template that can be introduce specific mutations

Conditional mutagenesis

About 30% of knock-outs are lethal, genes often have functions in different tissues or at developmental stages that are required for viability

• limitations of knock-out strategy to desired tissue or developmental stage, does not cause ablation in other essential functions

• uses site-specific recombinase enzymes, that can be expressed in select tissues or stages

• alleles, containing recombinase recognition sites

Site-specific recombinases

Their cognate recognition sites, set the stage for conditional mutagenesis

• harvest from DNA phases

• setup recombination event for us

Following knock-in of a “flozed-allele” to a genomic locus

Cre recombinase can excise DNA between the loxP sites, disrupting the allele

• loxP sites are introduced by knock-in

• intro - loose loops or large groups

Tissue-specific expression of Cre recombinase leads to tissue-specific disruption of targeted locus

Cre-lox tissue-specific knockout

• heterozygous for GeneX conditional knockout after 1 generation

• how husbandry can be used to prepare unique targeting of floxed locus in mice

Generate homozygous knockouts

Additional crosses are needed to make a mouse that has two floxed alleles and Cre expression transgene

• results in null effect in liver

Control mice are needed to determine that transgenic cre cassete and the loxP sites 

Do not cause any phenotypic effects on their own

• A) homozygous loxP control - check they don’t have strange livers before hand

• B) cre transgene control - make sure cre is effective

• C) cre, floxed het control

One Copy of floxed gene in genotyping PCR (heterozygous for loxp gene)

Ear punch) has 3 bands: 250bp Alb-Cre, 300bp Gene X, and 400bp floxed gene X

Liver) has 3 bands: 100bp disrupted locus, 250bp Alb-Cre, and 300bp Gene X

Two copies of floxed gene in genotyping PCR

Ear punch) has 2 bands: 250bp Alb-Cre and 400bp floxed gene X

Liver) has 2 bands: 100bp disrupted locus and 250bp Alb-Cre

Why are Floxed alleles generally non-disruptive in the absence of recombinase activity?

Modest size of loxP sites permit integration into intronic or intergenic regions such that coding information or expression of the target gene is not altered

How is Cre expression limited to select tissues?

Cre expression can be controlled by tissue-specific promoter (or some regulatory element)

to limit its expression to desired regions

Gene Drive

Converting heterozygous to homozygous mutations

• can disrupt locus

• knockout homologous allele (ensure all alleles)

• Good for targeting reproductive system in misquito to kill them off