A7-A8

Question 1

Plasticity (Greek “plastikos” – to mold) A phenomenon used to describe a cell that can become a different
type of cell Lineage - descendants of a common ancestor (via cell division)
* Lineage restriction – cells in a lineage have a tendency to stay within that lineage
* Differentiation the process by which a less specialized cell becomes a more specialized cell type
* Determination cellular changes in genetic programming that will eventually induce a specific cell type.
* Cells often become determined for a future role long before they differentiate
* Commitment when the determined state of the cell becomes (largely) irreversible
* Progenitor Cell – Generic term for any cell with the capacity to divide/differentiate that is more primitive (less
differentiated) than daughter cells in the lineage.
* Lineage Reprogramming (see Transdifferentiation)
* Transdifferentation when a somatic cell transforms into another somatic cell type without an intermediate
pluripotent state or progenitor cell type - a type of metaplasia
* Transdetermination - a switch in lineage commitment in a stem or progenitor cell to a closely related cell type
–often requires cell-cell coordination and communication to differentiate new tissue type
* Lineage Priming - a molecular model of stem cell (SC) differentiation in which proliferating SCs express a
subset of genes associated to the differentiation pathways to which they can commit (e.g. HSCs)
* Programming – The pattern of gene expression that induces commitment to a specific cell type in a l

Question 2

Extrinsic signals can induce a change in gene expression back to a more primitive state (de-differentiation).
* Extrinsic signals can induce a change in gene expression defining one type of differentiated cell to another.
* Transdifferentiation can occur via a relatively more primitive intermediate (dedifferentaion/redifferentation).

Question 3

Genomic DNA is not often exposed, it is packed in higherorder protein complexes (chromatin).
* During M-phase the replicated DNA (from S-phase) is
most packed into ‘chromosomes’ with the two sister
‘chromatids’ held together at the centromere.
* The centromere attaches to the spindle during M-phase to
facilitate movement of sister chromatids to each pole
during telophase

Question 4

Transcription factor [Activator] – promotes transcription by RNA polymerase
* Transcription factor [Repressor] – blocks transcription by RNA polymerase
* TFIID – a subunit of RNA polymerase II – the main protein complex that transcribes mRNA in eukaryotic cells.
* HDAC – Removes acetyl groups from histone causing a closed (inaccessible) chromatin structure.

Question 5

Genes can be ‘active’
transcribing RNA or
not.
* This is regulated at
several levels.

Question 6

‘Master’ or lineage-specifying transcription factors can
coordinate reprogramming one cell type to another.
* The initial target of these master TFs are usually other
TFs that are lineage specific.
* TFs usually bind a specific sequence of DNA

Question 7

MyoD (Myoblast Determination protein) the first
lineage defining TF, discovered in 1987.
* Transgenic expression of MyoD in fibroblasts
transdifferentiates them into skeletal muscle.
* GATA1 - GATA Binding Protein 1 - regulates HSC
differentiation.
* C/EBP α - CCAAT/enhancer-binding protein α –
regulates HSC differentiation.

Question 8

MyoD is regulated by
hypophosphorylated RB
acting as a co-activator of
myoblast differentiation
genes

Question 9

• The timing of transcription factor activity
  plays a role in programming cell lineage

Question 10

• C/EBPα - CCAAT-enhancer-binding protein α
• A hematopoiesis and granulopoiesis
  transcription factor that induces myeloid
  lineage-specific genes and regulates the
  cell cycle
• GATA2 - GATA Binding Protein 2
• A transcription factor that promotes
  hematopoietic cell differentiation

Question 11

The Waddington “landscape” of cell fate decisions and the
master transcription factors for each.

Question 12

During normal differentiation, more primitive cells move ‘downhill’
(metaphorically) in a lineage forming more lineage-restricted precursors
until they reach a differentiated state.
* Differentiation causes cells to move ‘uphill’ to a more primitive state. The
uphill metaphor implies that this is more difficult than progressing normally
along the differentiation lineage.
* Transdifferentiation is characterized as moving from one ‘valley’ to another,
rather than climbing back ‘uphill’ to a more primitive state.

Question 13

Transcription factors almost always work as multiprotein complexes
* Rb can repress genes involved in differentiation
* Transcriptional feedback loops (positive/negative)
can reinforce a pattern of gene expression.

Question 14

• PU.1 - Purine Rich Box-1 / SFFV Proviral Integration
  Site-1 (Spi-1)
• Transcription factor that induces genes needed
  in immune/hematopotetic lineages
• GATA1 - GATA Binding Protein 1
• Transcription factor that induces development
  of erythrocytes and platelets (megakaryocytes)
• CBP – Cyclic adenosine
  monophosphate Response Element Binding
  protein Binding Protein
• Transcriptional co-activator

Question 15

Reprogramming

Answer 15

a.
‘Pioneer
’ transcription factors
can access closed chromatin
.
b. Reprogramming factors
recruit other factors and work
cooperatively to activate or
inhibit target gene
expression.
c. Reprogramming factors
could refine the binding
profile of other
reprogramming factors
during direct reprogramming.
The expression of a single
reprogramming factor may
induce the expression of
lineage genes non
-specific to
the target cell type.

Question 16

Remember Oct4 and Sox2 & iPSCs?
* Nucleosome depleted regions (NDRs) at
active promoters and enhancers are
occupied by transcription factors and
chromatin remodelers.
* When these are removed— for example,
during differentiation — there is now
increased nucleosomes at the regulatory
region and DNA methylation.
* DNA methylation stabilizes silent state,
promoting epigenetic inheritance during cell
division.
* OCT4 targets its own promoter to maintain
expression.

Question 17

Octamer-binding transcription
factor 4 (OCT4)
* A transcription factor that
activates genes that keep
embryonic stem cells in a
pluripotent state.
* Sex determining region Y-box 2
(SOX2)
* Interacts with OCT4 to
activate pluripotent gene
expression and repress
genes involved in
differentiation

Question 18

During the process of
embryonic stem cell
differentiation pluripotency
and developmental
regulatory (differentiation)
are suppressed and
somatic genes are
activated.
* When an iPSC fate is
induced, pluripotency genes
are re-activated.
* However, a problem
with some iPSCs is
that development and
somatic gene
expression is not fully
repressed like in ESCs.

Question 19

Remodeler
– proteins that modify
the spacing or organization of
nucleosomes (DNA wrapped around
an octamer of histone proteins).
Reader
– a protein that senses
modifications to the exposed ‘tail’
region of histones.
Writer
– an enzyme that modifies
histone tails.
Eraser
– an enzyme that removes
modifications from histone tails.
Writer
– an enzyme that adds
methyl groups to C or A bases.
Eraser
– an enzyme that removes
methyl groups from C or A bases.
Reader
– a protein that senses
methyl
-C or methyl
-A bases

Question 20

DNA methylation

Answer 20

Adding methyl groups to adenine (A)
and cytosine (C) is a reversible process
that can change gene activity.
* Methylation is usually repressive.
* DNA methyltransferases add methyl
groups to cytosine
* Demethylases remove methyl groups.

Question 21

The cycle of DNA
methylation during
division/differentiation

Answer 21

• During early embryogenesis, DNA is
  mostly unmethylated (top left).
• As cells divide / differentiate genes
  acquire DNA methylation (red circles) by
  DNMT3A DNMT3B (top).
• Methylation of CpG islands silences
  genes. Methyl-binding proteins chase
  histone H3K9 to be deaceylated then
  methylated, recruiting heterochromatin
  protein 1 (HP1) resulting in closed
  chromatin (bottom right).
• At S-phase, newly synthesized DNA (in
  green) is unmethylated and the marks
  must be reestablished on opposite the
  existing marks.
• Adult methylation patterns are erased by
  epigenetic reprogramming during
  embryogenesis (top left).

Question 22

Heterochromatin protein 1 (HP1) – recruits DNA methyltransferases
* DNMT – DNA methyltransferase 1 –deposits methyl groups on newly
synthesized DNA opposite exiting marks maintaining silencing.

Question 23

• H3K27 tri-methylation (H3K27Me3) is a * H3K27Me3 repressive mark.
• H3K4 tri-methylation (H3K4Me3) is an
  activating mark.
• Both modifications can be present. In this
  case the gene is considered ‘poised’ for
  transcription.

Answer 23

Histone subunit 3 (H3) – Lysine (K) amino
acids within the N-terminal ‘tail’ can be
modified by varying (mono, di, tri-) addition of
methyl groups.

Question 24

Acetylation of the tail OF histones H2B and H4 inhibits the folding of nucleosome arrays into secondary and
tertiary chromatin structures allowing access to transcription factors and other transcription co-activators.

Question 25

KMTs = histone–lysine Nmethyltransferases

Answer 25

* Model * KMT2C and KMT2D act at distal enhancers * KMT2F and KMT2G are the major enzymes at gene promoters. * KMT2A and KMT2B function at both regulatory regions. * Recruitment of KMT2s to different genomic regions is facilitated by interactions with transcription factors or RNA polymerase II (RNAPII).

Question 26

KDMs = histone–lysine demethylases

Answer 26

* During differentiation, KDM5A represses factors that drive cell cycle (e.g. mitogens) and promotes cell cycle exit. * KDM5B inhibits cell cycle exit, by repressing expression of cell cycle inhibitors (e.g. p27) and several differentiation markers.

Question 27

CPG islands

Answer 27

* 60% of human genes have CpG islands (CGIs) at their promoters and frequently have nucleosome -depleted regions (NDRs) at the transcriptional start site (TSS) . * Nucleosomes flanking an ‘active’ TSS are marked by trimethylation of lysine 4 within histone at (H3K4me3), and the histone variant H2A.Z, which is antagonistic to DNA methyltransferases (DNMTs). * Downstream of the TSS, DNA is mostly CpG - depleted and methylated. CGIs, which are sometimes located in gene bodies, mostly remain unmethylated. * LMR, low -methylated region.

Question 28

epigenetics

Answer 28

Hypermethylation of CpG islands in promoter regions by DNMTs inhibits gene expression (1). * Histones are protein octamers consisting of a pair of each of four core proteins, histone protein 2A (H2A), H2B, H3 and H4. * Acetyl groups (AC, filled red triangles) are placed by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs) * Acetylation weakens chromatin compaction status making DNA accessible to transcription factors (2). * Histone methylation is regulated by methyltransferases (HMTs) and demethylases (HDMs); methyl groups (filled blue rectangles) on histone tails recruit proteins that repress or increase gene expression (2). For instance, methylation of lysine 27 (K27) on the tail of H3 can repress gene expression.

Question 29

Bmi-1 regulates HSCs

Answer 29

Bmi-1 was isolated as an oncogene * Is now a transcriptional repressor belonging to the Polycomb group (PcG) of proteins. * A mouse Bmi-1 knockout: * Neurological defects * Severe proliferation defects in lymphoid cells * Bmi-1 is responsible for both HSC and neural stem cell function * Bmi-1 * RING (Really Interesting New Gene domain) – Mediates DNA or protein binding * HTH (Helix – turn –helix domain) DNA binding domain * PEST (Proline Glutamate Serine Threonine rich domain) – Promotes Polyubiquitination by E3 ligases

Question 30

Bmi-1 & the cell cycle

Answer 30

BMI1 promotes cell proliferation by directly or indirectly inhibiting the transcription of CDKN2A, which encodes p16INK4A (also known as p16) p14ARF .

Question 31

Bmi-1/is a part of the ‘Polycomb repressive complex (PRC1)’

Answer 31

* Bmi-1 is NOT a transcription factor! * The PRC1 is a complex of proteins that modifies chromatin (DNA + protein) that has a direct influence on whether transcription factors can bind to DNA. * This provides long-term transcriptional memory at CKI genes etc.

Question 32

Transcriptional memory - Polycomb proteins

Answer 32

Polycomb complex (PcG) proteins associate with chromatin and repress transcription by RNA polymerase II. * PcG recognizes specific genes, and spreads along the chromatin to inhibit transcription

Question 33

PcG - discovery

Answer 33

* Bithorax Complex (BX-C) a region of homeobox (Hox) genes, including Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B). * This region has complex and overlapping gene regulatory elements including the abx/bx, bxd/pbx and iab elements. Parasegment (PS) – a region containing multiple cells within the embryo that follows the segmented structure. * LacZ- β-galactosidase gene from E. coli, not normally expressed in flies. * PRE – Polycomb response element (a DNA sequence recognized by PcG)

Question 34

PcG preserves gene expression patterns as embryo cell divide/differentiate

Answer 34

a) The Drosophila melanogaster larva is derived from the embryonic parasegments. Imaginal discs originate from specific pairs of embryonic parasegments and later give rise to adult structures. b) The Bithorax complex (BX-C) also contains imaginal disc enhancers. However, these enhancers do not contain positional information. The Bithoraxoid (bxd) regulatory region drives expression (dark grey) in all discs. c) Addition of the bxd Polycomb/Trithorax response element (PRE) (from the same regulatory domain) leads to silencing in all discs (light grey). d) If the correct embryonic enhancer is added to the construct together with the PRE, the imaginal disc enhancer becomes restricted to the correct expression domain, with the boundary at parasegment Thus, the bxd PRE transfers positional information from the early embryonic enhancers to the late enhancers.

Question 35

There are two different Polycomb repressive complexes (PRCs)

Answer 35

Polycomb repressive complex 1 (PRC1) * RING1A and RING1B are E3 ligases that guide the transfer of a Ub onto H2AK119 (H2AK119ub1). The Polycomb group RING finger (PCGF) subunit regulates PRC1 targeting to chromatin. PRC2 * Enhancer of zeste homolog 1/2 EZH1/2 of PRC2 act as H3K27 methyltransferases. The Embryonic Ectoderm (EED), Supressor of zeste 12 (SUZ12, and RGB Associated Proteins 46/48 (RBAP46/8) guide the methyltransferase to H3.

Question 36

H2K119 ubiquitylation and repression

Answer 36

* PRC1monoubiquitylation of histone H2A at K119 (H2AK119ub) recruits PRC2that trimethylates histone H3 at K27. H2AK119ub and H3K27me3 both repress transcription. * Deubiquitylation of H2A, which can be carried out by deubiquitinases (DUBs), accordingly activates transcription.

Question 37

Models of how PRC recruitment maintain gene expression

Answer 37

Instructive model - newly recruited Polycomb complexes lead to Polycomb chromatin domain formation, which then directs repression of transcription by RNA polymerase II (Pol II) at the associated gene. b) Responsive model - Polycomb complexes constantly ‘sample’ chromatin at regulatory elements in order to respond to the transcriptional state of the associated gene. Reduced transcription would allow establishment of Polycomb chromatin domains that protect against reactivation. When genes are actively transcribing, the presence of Pol II would block establishment of Polycomb domains

Question 38

Trithorax complexes (TrxG) act antagonistically to PcG

Answer 38

Modify histones and modify chromatin. * COMPASS family proteins * SET1, Trx, Trx-related, MLL * Histone-lysine Nmethyltransferase * COMPASS & COMPASS-like complexes are capable of monomethylation, demethylation. * Menin – a ‘scaffold’ protein * Grey - common subunits * Green - shared subunits * (you don’t need to know specific names).

Question 39

Trx (MLL) levels change with the cell cycle

Answer 39

* SCF/SKP2 and APC/CDC20 target MLL for degradation during S phase and mitosis, respectively. * During G1, MLL1 or MLL2 associate with E2F to control G1 phase cell cycle genes. * During mitosis, the MLL complex is retained on chromatin, which could facilitate the inheritance of active gene expression during cell division.

Question 40

Maintenance of gene activity by PcG and TrxG

Answer 40

PRE – PcG recruiting element TRE – TrX recruiting element

Question 41

How are chromatin signatures are stably transmitted during cell division?

Answer 41

Histones at Trithorax group (TrxGMLL1) and Polycomb group (PcG) binding sites are exchanged multiple times during cell division. * Local concentration of PcG proteins bind/rebinds with H3K27me3 after Sphase to reestablish gene repression. * Pc (part of PRC2) also ‘writes’ H3K27me3. * MLL1 likely remains associated with condensed mitotic chromosomes.

Question 42

Epigenetic regulation of haematopoietic stem cells

Answer 42

ES cell cycle is shorter relative to other cells (∼11–16 hours vs. ∼24 hours) due to an abbreviated G1 phase. * in ES cells, cyclin E–CDK2 (E/2) is constitutively active throughout the cell cycle, which allows the transition of ES cells from M phase directly to late G1 * Upon ES cell commitment, the cell-cycle length is extended as cyclin E–CDK2 activity comes under the control of cyclin D–CDK4 and phosphorylated RB. * CDK activity: +/-, negligible; +, low; ++, intermediate; +++, high.

Question 43

Stem cells – the ‘barrel model’ of the balance molecular ‘forces’ between ‘commitment’ and proliferation.

Answer 43

Early G1 * Mitogen-dependent cyclin D–CDK4 stimulation is represented by taps into the Proliferation barrel. * When RB hyperphosphorylation and cyclin E–CDK2 activation reaches the R point (Proliferation barrel is full), cell-cycle progression occurs. * Certain proliferative stimuli (e.g. MAPK) induce developmental commitment and proliferation (represented by a tap into both barrels).

Question 44

PcG / TrxG bi - stable occupation of gene promoters in stem cells

Answer 44

Self -renewing stem cell daughters have bi -stable regulation of genes encoding master transcription factors that specific linage etc. * Early in the commitment process (induced by MAPK signaling), PcG is removed from genes directing both lineages. * As cells differentiate, PcG is reestablished at genes not needed for the chosen lineage . * Trx remains at genes that drive differ enation along a particular lineage

Question 45

How niche signals affect stem cell gene regulation

Answer 45

Niche is composed of local cell populations and the secreted and cell -surface -bound molecules these cells generate as well as the local metabolic state of the niche (e.g. oxygen tension. Important regulators of stem cell function are: morphogens (Morph - e.g. Notch, Wnt and Hedgehog), cell –cell and cell –extracellular matrix (ECM) adhesion molecules (e.g. cadherins and integrins) and hypoxia. These factors can alter the balance between commitment and proliferation by: a) Affecting entry into the cell cycle. b) Stimulating proliferation. c) Blocking expression of development by recruitment of PcG .

Question 46

Chromatin bivalency

Answer 46

Bivalent genes are poised to rapidly increase / decrease their transcription rate. * This drives rapid gene expression transitions as stem cells differentiate, * This occurs by local competition between PRC1/PRC2 and Trx proteins at CpG islands (CGI) * This would provide a binary response to graded activation signal(s ) .

Question 47

How to determine if proteins interact with DNA?

Answer 47

ChIP – Chromatin immunoprecpipatio n * CUT&RUN – antibodies are bound to a micrococcal nuclease which cleaves the DNA surrounding the protein, freeing it for extraction * CUT&Tag – antibodies are bound to a Tn5 transposable element enzyme which inserts small DNA sequences adjacent to the protein of interest. Tn5 elements are used for PCR to selectively amplify the DNA that was bound by the transcription factor (TF) of interest.

Question 48

Bivalent domains result from Polycomb and Trithorax activity at bivalent CpG islands (CGIs)

Question 49

Most genes in a newly fertilized zygote are NOT transcribed – with bivalent histones present. * KMT2B, PRC1 and PRC2 already present in the oocyte form H3K4me3, H3K27me3 H2AK119ub domains to repress differentiation gene expression. * As transcription starts at zygotic genome activation (ZGA) an initial pattern of pluripotent genes become activated via KDM5A, KDM1 and KMT2.

Question 50

Variant PRC1 (vPRC1) nucleates H3K27me3 in pre -implantation embryos. * Canonical PRC1 (cPRC1) ‘spreads’ H3K27me3. * Bivalency occurs at promoters of developmental regulatory genes post -implantation

Question 51

No matter what type of reprogramming, SCNT, iPS cells or ES cell mediated, a passage through the cell cycle improves the process.

Question 52

Pausing the cell cycle can reversible (quiescent) or irreversible (senescent and differentiated). * Senescent cells are dysfunctional cells that no longer proliferate and are permanently withdrawn from the cell cycle. * Differentiated cells are withdrawn from the cell cycle due to specializations that are incompatible with division.

Question 53

Cells are no longer able to enter the cell cycle The Hallmarks of the Senescence Senescent cells exhibit the following four interdependent hallmarks: (1) cell-cycle withdrawal (2) macromolecular damage (3) secretory phenotype (SASP) (4) deregulated metabolism

Question 54

NF-κB Effector of the immune signalling * CEPBβ Effector of immune / inflammatory response * GATA4 Effector of inflammatory response in response to NF-κB

Question 55

Senescence activates p16 and p14/ARF

Answer 55

* Senescence -inducing signals, including those that trigger a DNA -damage response usually engage either the p53 or the p16 –pRB tumour suppressor pathways. * Senescence signals that engage the p16 –pRB pathway generally do so by inducing the expression of p16, another CDK inhibitor that prevents pRB phosphorylation and inactivation.

Question 56

Causes of senescence

Answer 56

Extensive DNA damage response (DDR) causes senescence, via p53 activation. Minor DDR at telomeres is sufficient to trigger senescence. Oncogene activation induces hyperproliferation and altered DNA replication patterns that ultimately result in replication stress and DNA damage accumulation at fragile sites, including telomeres. Besides DDR, senescence features include upregulation of p21 and p16, increased ROS, metabolic changes like (SA - β -gal) and a senescence - associated secretory phenotype (SASP).

Question 57

Senescent cells execute distinct biological functions, which can have deleterious or beneficial consequences in a contextdependent manner. Senescent cells also promote reprograming to an embryonic state, at least partially through IL6. The reprograming, on one hand, can support tissue regeneration and, on the other hand, favours tumour development.

Answer 57

Interleukins (IL) are cytokines ‘cyto/cell + kinos/movement = attract cells, that are expressed in response to cell damage. * Matrix metalloproteinases (MMPs ) break down the extracellular matrix * GROα is a chemokine – a chemical attractant for immune cells * IFNγ – Interferon gamma – signals induce senescence in receiving cell

Question 58

SASP

Answer 58

Multiple stimuli provoke senescence and SASP. * Irreversible cell-cycle arrest triggered by severe DNA damage causes SASP in senescent cells. * Note that overexpression of p16 or p21 causes growth arrest, but not SASP. * SASP cells secrete high levels of inflammatory cytokines, immune modulators, growth factors, and proteases. SASP cells may also produce exosomes and ectosomes containing enzymes, microRNA, DNA fragments, and other bioactive factors. SASP cells are initially immunosuppressive but progress to become proinflammatory.

Question 59

Transient exposure to SASP promotes plasticity and increases iPSC reprogramming in neighboring cells, promoting tissue repair and regeneration. * Chronic exposure to SASP cells activates a cell-intrinsic senescence which is thought to mediate tumor suppression and/or aging.

Question 60

* Werner syndrome (WS) and Hutchinson -Gilford progeria syndromes (HGPS) lead to an accelerated aging phenotype. * MSCs derived from ESCs isolated from patients with mutations in the the Werner Syndrome Helicase (WRN) gene had excess DNA damage and change in overall nuclear architecture consistent with altered programming.

Question 61

Age-related changes in the cells and molecules composing the stem cell niche lead to aberrant stimulation of stem cells. * This affects gene programming affecting quiescence, metabolism and differentiation. * Extrinsic signals also drive epigenetic changes that can lead to inappropriate activation of developmental pathways.

Question 62

The proliferation rate of a stem cell affects its exposure and responses to mechanisms of aging. Although quiescent stem cells are less susceptible to DNA damage, when it does occur, they are more likely to utilize error-prone nonhomologous end-joining (NHEJ) as a repair mechanism, whereas cycling stem cells are more likely to use homologous recombination (HR). * Cycling stem cells also have higher metabolic demands than quiescent stem cells.