Cellular differentiation and reprogramming

• Principles of cell-substratum (ECM) interactions

  • Differentiation is the process by which cells specialize into different cell types
  • Chromatin can be open or closed
  • Generally, chromatin closure is associated with differentiation
  • The histone code helps organize chromatin and regulate gene expression
• In cell division, polymerase II leaves the DNA and stops transcribing genes – how does the cell remember what genes to turn back on?


  ‘Bookmarks’ are a combination of epigenetic marks and protein

  partners that leave instructions on the genes

  • Bookmarks can be reprogrammed by the micro-environment, and in cancer
  • The microenvironment can also control developmental decisions and cell identity
  • Bookmarks can mark which genes are paternal or maternal
• Levels of chromatin organization

  • Chromatin that remains tightly compacted after mitosis is called heterochromatin
  • Euchromatin returns to a dispersed, active state after mitosis.
• Nucleosomes: The Lowest Level of Chromosome Organization

  Chromosomes consist of:

  • chromatin fibers, composed of DNA and associated proteins
  • histones, a group of highly conserved proteins
  • DNA and histones are organized into repeating subunits called nucleosomes
  • Histone H1 which acts as a linker for each nucleosome
• Nucleosome

  DNA wrapped around the core complex

  • complex has 2 of each:
  • H2A
  • H2B
  • H3
  • H4
  • forming an octamer
• histones

  Each histone has a flexible amino terminal tail that sticks out past the DNA
• Histone code

  histone tail modifications can serve as docking sites to recruit nonhistone proteins to open or close chromatin and alter the way histones of neighboring nucleosomes interact.

  Modifications:

  • phosphorylation
  • acetylation
  • methylation
  • ubiquitination

  The histone code hypothesis states that the activity of a chromatin

  region depends on the degree of chemical modification of histone

  tails.
• DNA and Histone methylation

  DNA: A methyl group is added to the 5-carbon position of cytosine residues in DNA by DNA methyl-transferases.

  Histones: Histones are methylated on arginine and lysine residues(mono- or di-) by histone methyl-transferases
• Histones can be acetylated on lysines

  HAT – histone acetyl-transferases

  HDAC- histone deacetylases

  • HAT leads to open chromatin
  • HDAC or histone methyltransferases lead to closed chromatin
• histone methyltransferases lead to closed chromatin
• What does heterochromatin protein 1 (HP-1) do and how is it recruited?

  lysine 9 of histone tail recruits HP-1 protein which contributes to heterochromatin (closed chromatin).

  • if H3K9 is methylated: HP-1 binds and chromatin is turned off
  • If H3K9 is acetylated: HP-1 doesn't bind and chromatin is on
• Heterochromatin Protein-1

  • Heterochromatin protein- 1 (HP1) can bind to methylated lysine 9 of histone 3
  • The modification and HP1 are associated with closed chromatin
  • HP1 contains a chromodomain which binds methylated histone
• Chromodomain

  area of proteins that will bind to methylated amino acids / histones
• How does a cell know which genes were ‘on’ before the cell cycle interrupted transcription?

  When polymerase II falls off during mitosis (because of chromatin compaction many proteins fall off and transcription stops) how does the polymerase know which genes were turned on so it can go back to transcribing them.

  • H3K27ac doesn't fall off during mitosis and acts as a bookmark (epigenetic mark) for transcription when interrupted during the cell cycle.
  • Considered 'short-term cell memory' since only relevant to mitosis
• What silences a whole chromosome?

  Although cells of females contain two X chromosomes, only one of them is transcriptionally active. The other X chromosome remains condensed as a heterochromatic clump called a Barr body.

  • The RNA known as Xist is noncoding and when transcribed binds to the chromatin to compact the DNA so most genes cannot be transcribed
  • After compaction by Xist, histone methylation occurs that may recruit HP-1 and other proteins to close the chromatin
  • Xist remains the only active gene on the chromosome to promote the inactive state
• The mammary gland microenvironment can

  reprogram cells of other tissues

  • The epithelial lineage cells of the mammary gland are removed,

  leaving behind a cleared microenvironment. Mammary cells replaced

  with:

  • Testicular cells
  • Neural stem cells
  • Embryonic stem cells
  • And from each of these, a functional mammary gland forms that can produce milk!
• What cells have the most closed chromatin?

  Tissue-specific cells
• What cells have the most open chromatin?

  Stem cells
• Principles of cell-substratum (ECM) interactions

  • Every tissue likely has a tissue specific stem cell for repair and

  maintenance

  • Only the zygote is totipotent, the embryonic stem cell is pluripotent, the tissue specific stem cell is multi-potent
• What does multipotent mean and what cells are multipotent?

  adult stem cells are multipotent and means that multiple cells of that specific tissue can be made
• What does pluripotent mean and what cells are pluripotent?

  Embryonic stem cells are pluripotent and can produce all the required material but not the amniotic sac or the placenta
• What is totipotent and what cells are totipotent?

  The zygote is totipotent and can form everything required for life
• What is the procedure of therapeutic cloning ?

  1. Removal of somatic cells
  2. nucleus donation from somatic cells to enucleated oocyte (nucleus removed)
  3. allow the blastocyst to develop; inner mass of blastocyst contains embryonic stem cells
  4. Induce embryonic stem cells to differentiate
  5. Transplant differentiated cells back into patient
• Stem cells are undifferentiated cells that are capable of ...

  1. self-renewal: the production stem cells with a similar capacity to proliferate and differentiate
  2. commitment: production of cells committed to differentiate whereby they produce progenitor cells that will differentiate.

  Stem cells can either divide to make another stem cell (self-renewal) or a progenitor (commitment).
• What does unipotent mean?

  These are differentiated cells that can only make one type of cell within a specific tissue
• Epigenetic states of cells at different stages of development?

  1. Global DNA demethylation: blank canvas, all chromatin is open
  2. global repression of differentiation: repression of multiple lineages
  3. Repression of lineage-specific genes
  4. Promoter hypermethylation and de-repression of lineage genes
• The egg can reprogram DNA

  Instead of histones, sperm have protamines

  • The protamines act like histones and are very easily removed from the DNA
  • Protamines are joined by disulfide bonds that are reduced by Glutathione in the egg, causing the sperm chromatin to uncoil and be reprogramed
  • This is followed by histone acetylation and DNA demethylation
• Cloning is asking cells to reprogram their DNA, based on what property of the cell is this based on?

  Plasticity: The ability of a partially differentiated adult stem cell to change its genetic program and differentiate into cells of another tissue type
• What in our bodies is capable of reprogramming the differentiated state of chromatin

  • the microenvironment
  • the egg
• can humans force differentiated cells to become stem cells?

  Scientists can create stems cell by forcing the expression of 3-4 pluripotent genes responsible for pluripotency changes from an adult stem cell to a stem cell. This makes an induced pluripotent stem cell (iPSC)