Differentiation, gene expression and cellular memory

1
Lecture 7

• Differentiation, gene expression and cellular
  memory

2
Differentiation

• How do multiple cell types arise from single
  cell?
• all cells have same genome (genomic equivalence
  or constancy)
• genes can be differentially expressed

3
what controls cellular phenotype?

• Where are the genetic factors? (Mendel posited
  form-building-elements of unknown location)
• Roux, Boveri, Wilson a cells phenotype is
  controlled by nucleus
• only protozoan cell fragments containing nucleus
  can regenerate
• T.H. Morgan phenotype controlled by cytoplasm
• removal of cytoplasm can affect phenotype

4
genes are in the nucleus

• fly genetics
• sex chromosomes and sex-linked traits (white)in
  Drosophila (Morgan, 1911)
• geneticists nuclei must control cell fate
• embryologists then how come cells are different?

5
cytoplasm can affect gene expression

• cell fusion (heterokaryon) experiments

Fig 9.31
6
phenotype of cell (i.e. gene expression) involves
nucleocytoplasmic interaction

• Both are important
• nucleus can respond differently to different
  cytoplasmic signals
• so cell differentiation could reflect differences
  in cytoplasm even if nuclei the same
• but do all cells in organism have same DNA?
  (genomic constancy) largely yes--evidence next
  lecture

7
Differentiation simple models

• Naegleria
• simplest. single cell can adopt either of two
  states
• Volvox
• Soma/germline distinction
• Dictyostelium
• Multiple cell types in slug

8
Naegleria gruberis quick-change act

• Single celled protozoan
• Amoeboid in abundant food
• Develops flagellae if starved
• (Amoeboflagellate
• Takes 90 minutes to change
• Block change by Actinomycin D
• So RNA synthesis required
• Tubulin gene

9
Volvox carteri and the division of labor

• Two cell types
• 2000 Somatic
• 16 Germline (gonidia)
• Asymmetric division gives 1 somatic 1 gonidial
  daughter
• regA loss of function mutant no somatic cells
• regA gain of function mutant no germline

10
Control of soma/germline distinction by RegA

• Two kinds of mutation
• regA loss of function mutant no somatic cells
• regA gain of function mutant no germline
• Cloned nuclear protein

11
Differentiation in Dicty

• Amoebae all look the same
• Aggregate into mound (Figs 8.38-40) cAMP
  attracts
• Slugs contain two major cell types, prespore and
  prestalk

12
The slug contains multiple cell types

• two major cell types, prespore (Psp, gray) and
  prestalk (Pst--the rest)--Fig 6.24,25

13
Prespore vs. prestalk

• Prestalk cells (green) aggregate in center and
  make cAMP (relay mechanism)
• So 2 cell types already at this stage

14
What makes cells become prestalk or prespore?

• Amoebae are not all the same--choice depends on
  stage in cell cycle that they receive cAMP signal
• Signal in S or early G2--become prespore
• Signal in late G2--become prestalk
• cAMP signal transduction pathway resembles the
  Wnt pathway..

15
Dynamics
GFP-labeled Pst cells in slug and culmination

• Movies from the online Dictyostelium tutorial
• http//dictybase.org/tutorial/

16
regulation of eukaryotic gene expn

• Multiple levels, regulation at any
• transcription
• splicing
• export
• mRNA localization/stability
• translational
• post-translational
• most widespread
• (4-5) in early embryos more later

17
transcriptional control

• Basal transcription machinery
• binds promoter sequence
• TATA binding factor etc
• directly recruits RNA polymerase
• same in all cells
• transcriptional regulatory proteins
• bind enhancers if activating
• action at a distance
• indirectly recruit basal machinery
• factors cell type specific

18
enhancers

• definition vague, but any region of DNA that
  influence gene transcription that is not part of
  promoter
• can be close in (100 bp) or 10s of kb away
• 5 or 3 to gene
• act in either orientation
• simple or complex multiple binding sites allow
  combinatorial control

19
transcriptional regulators

• usually loosely called transcription factors, but
  not to be confused with basal TFs

Fig 9.3
20
Transcriptional regulators

• general structure
• DNA binding domain
• activation domain
• other regulatory domains
• large families of proteins sharing similar DNA
  binding domains
• homeodomain family
• T box, Zinc finger, bHLH, etc

21
Brachyury

• named after the mouse mutant (short tails)
• cloned in 1990, protein found in nucleus binds
  DNA
• DNA binding site is palindrome, suggesting dimer
• protein has a T domain (200 amino acids)
  encoded by a T box in the DNA

22
what determines specificity?

• i.e. why dont all T-box proteins activate all
  genes with T-box binding sites
• answer lies in details of protein-DNA
  interactions
• single base changes in DNA binding site

23
example Pit-1 and pituitary cell types

• Pituitary gland contains 6 distinct cell types
• 3 express the same transcription factor, Pit-1,
  but do not express the same hormones
• GH-expressing cells (somatotropes)
• prolactin expressing cells (lactotropes)
• Scully et al 2000. Examine how Pit-1 interacts
  with enhancers in GH gene versus prolactin gene

24
How inductive signals affect transcription

• signal transduction pathways
• from cell surface receptor to usually a
  transcription factor in nucleus
• TGFb signaling as example
• Ligand TGFb binds dimer of Type I/TypeII
  receptors
• Type I is kinase
• Phosporylates cytoplasmic domain of type II
• Creating a binding site for Smad proteins
• Smads get phosphorylated and then form dimers
  with co-Smad proteins
• Translocate to nucleus and bind DNA (Smad Binding
  Elements)

Fig 3.34
25
Steroid hormone interact with receptors in cytosol
estrogen, testosterone, etc..
Fig 9.6
26
summary

• gene expression regulated at transcriptional
  level in most cases
• exception being the very early embryo before
  zygote begins transcription translational
  control rules there
• in embryos localization of determinants or
  localized signals both lead to localized txn
• but determinants, signals are transient. How are
  gene expression patterns maintained for long
  term?

27
cellular memory phenomena

• stability of differentiated state
• determination (of undifferentiated cells)
• stable patterns of gene expression over long
  periods,not affected by cell divisions.

28
Cellular memory mechanisms

• Tissue memory
• examples when we get to Drosophila segmentation
• epigenetic programming of gene expression
• positive feedback loop on transcription
• heritable changes in chromatin structure
• heritable DNA modifications (but not mutation)
• epigenetics any more or less stable
  alteration of gene activity that does not involve
  DNA mutation

29
transcriptional feedback loops

• W Fig 9.8

30
chromatin

• 2 meters of DNA in every human cell
• Packaging into chromatin
• Very roughly in two states
• Open euchromatin, genes available for
  transcription
• Closed heterochromatin

31
Histone tails are extensively modified

• Acetylation, methylation, phosphorylation
• Histones not boring any more

32
the histone code hypothesis

• Acetylation open, decondensed (removes ve
  charge on lysine residues)
• Thus histone acetyltransferases (HATs) open
  chromatin
• Histone deacetylases (HDACs) close it
  downroughly
• Methylation closed, condensed
• Combinatorial code? (David Allis et al 2000)

33
chromatin remodeling complexes

• Enzymes that catalyze nucleosome movement
• Large multiprotein complexes
• Trithorax (Trx) group generally activating
• Proteins contain domains that bind modified
  histone tails
• bromodomain proteins bind acetylated histones
• I.e. recognize open chromatin and open it further?

34
Anti-remodeling complexes

• Enzymes that prevent nucleosome movement, cause
  chromatin condensation
• Large multiprotein complexes
• Polycomb group (PcG) generally repressive
• Some complexes contain HDAcs

A DNA nucleosomes
Polycomb protein complex
EM of nucleosome arrays from Nicole Francis,
Harvard
35
epigenetic inheritance X-inactivation

• Tortoiseshell/calico cats are always female
• genotype O/o
• O/O cells are red
• o/o cells black
• X-linked
• mosaic of red and black is due to X-inactivation
  in XX heterozygote cats

36
X-inactivation

• inactive X highly condensed (heterochromatin)
  the Barr body
• heritable

Figs 9.9, 9.10
37
X-inactivation

• function is to equalize dosage of X-linked genes
  in XX and XY (dosage compensation)
• XXX cells inactivate 2 Xs--cell has a
  (mysterious) counting mechanism
• region of X, the X-inactivation center (Xic) is
  essential
• expresses 17 kb non-coding RNA (Xist) expressed
  by inactive X that acts in cis
• also its antisense transcript Tsix
• Xist initiates X-inactivation but not needed to
  maintain

38
The X-inactivation center (Xic)

• 50 kb region of X essential for inactivation
• expresses 17 kb non-coding RNA (Xist) expressed
  by inactive X that acts in cis
• also its antisense transcript Tsix
• Xist initiates X-inactivation but not needed to
  maintain

39
X-inactivation occurs in steps

• Kinetics studied in XX ES cells
• Xist sets in motion a series of chromatin
  inactivating steps resulting in transcriptional
  silencing 24h after Xist turns on
• PcG probably important
• Final step is DNA methylation

40
DNA methylation

• covalent modification of cytosine in some CpG
  dinucleotides
• inhibits transcription
• only in vertebrates
• pattern of methylation is inherited through cell
  division
• maintenance methyltransferase

41
why methylation state is heritable
Fig 9.11

• maintenance methylase recognizes
  half-methylated CpGs

42
Genomic imprinting

• in mammals only
• maternal and paternal genomes have different
  patterns of methylation
• a zygote derived from 2 maternal or 2 paternal
  genomes does not develop
• no parthenogenetic mammals (unless you block the
  imprinting system--Kono et al 2004)
• no obvious why
• an example of intragenomic conflict?