MCDB 436 (15): Epigenetic Modifications

human genome

98% shared with chimps
- human genome encodes for 30k genes, C elegans 20k (many human analogues)

organization of the genome

DNA (2nm) < nucleosome (11nm; histone core and DNA) < chromatin (30nm, 300nm, 700nm) < chromosome (1400nm)

types of epigenetic modifications

1) DNA methylation - at cytosine in CpG dinucleotide area (5mCpG)

2) histone modification - chromatin remodeling via post-translational modification of histones

DNA methylation

(for mammals); REVERSIBLE PROCESS

DNMT enzyme catalyzes the covalent attachment of a methyl group to the C5 position residues in CpG dinuc DNA sequences

introduced methylation patterns are preserved and maintained by DNMT1 during replication

DNMT1

preserve and maintain methylation patterns during replication

TET1-3

removes the epigenetic modification

DNMT3A/B

responsible for de novo methylation
- C5 position of cytosine residues in CpG dinucleotide DNA sequences

effect of DNA methylation on chromatin

results in condensed chromatin, leading to transcription inactivation

histone subunits and their products

H3 + H4 -> H3-H4 dimer -> H3-H4 tetramer

H2A + H2B -> H2A-H2B dimer ->

tetramer + dimer = histone octamer

histone protein structure

histone tail: can be acetylated, methylated, phosphorylated, or ubiquitinated

histone fold

four major types of histone modifications

histone acetylation - on Lys (K)

phosphorylation - on Ser/Thr (S/T)

methylation - on His/Lys/Arg (H/K/R)

ubiquitination

effects of histone modification on chromatin

modifications to histone tails render chromatin into open or closed conformation
- tighter wrapping = less accessible; looser wrapping = accessible DNA -> transcription

methyl = condensed
acetyl = loosened

histone methylation

catalyzed by histone methyltransferase enzyme (HMT)
- associated with activation OR repression of gene expression (depends on histone protein + AA residue)
- occurs on Arg, Lys, His

Lys methylation

monomethylated (me1), dimethylated (me2), or trimethylated (me3) on epsilon-amine group

Arg methylation

monomethylated (me1), symmetrically dimethylated (me2s), or asymmetrically dimethylated (me2a) on guanidinyl group

His methylation

monomethylated (me1)

histone methylation abbreviations

H3K4me3
- Histidine 3 methylated
- Lysine 4 trimethylated

H4K20me1
- histidine 4 methylated
- lysine 20 monomethylated

histone acetylation

dynamic process regulated by histone aceyltransferase (HAT) and histone deacetylase enzyme (HDAC)

HAT

RELAX chromatin -> promote interaction of RNA-polymerase and other TFs with DNA and activation of gene expression

(relax; take your hat off, stay a while)

HDAC

mediate CLOSED chromatin conformation -> downregulation of gene expression

(c = closed/condensed)

cell differentiation

a biological process wherein a cell develops and acquires a more specialized form and function
- changes may include cell shape, size, membrane potential, metabolic activities, responsiveness, etc
- changes brought about by modifications in gene expressions; crucial component of the cell differentiation process

differentiated cell

a cell that has changed in form and matured from being generalized into being more specific in terms of function

undifferentiated cell

a progenitor cell that is yet to undergo cellular differentiation

T cell differentiation

CD4 and CD8 T cells leave the thymus and enter circulation as resting cells in the G0 stage
- 2x as many CD4 than CD8

naive T cell activation/proliferation

characteristics: condensed chromatin, very little cytoplasm, and little transcriptional activity

can activate by recognizing Ag-MHC complex on APC/target cell
- IL-2 -> IL2R -> proliferation -> 48hrs, enlarges into blast cell and undergoes rounds of cell division -> effector or memory cells

transformation

conversion of a normal cell into a tumor cell
- changes at cellular, genetic, and epigenetic levels and abnormal cell division

6 hallmarks of cancer

1) sustaining proliferative signaling
2) evading growth suppressors
3) activating invasion and metastasis
4) enabling replicative immorality
5) inducing angiogenesis
6) resisting cell death

sustaining proliferative signaling

- the accelerator signals instruct cells to grow and divide chronically
- formation of oncogenes

oncogenes

genes that transform normal cells into cancer cells

discovery of Rous Sarcoma Virus

by peyton rous, nobel prize/physiology or medicine

chicken with sarcoma in breast muscle ->
remove sarcoma and break up into small chunks of tissue ->
grind up sarcoma with sand ->
collect filtrate that has passed through fine-pore filter ->
inject filtrate into young chicken ->
observe sarcoma in injected chicken

result of RSV discovery

RSV can transform infected cells into tumor cells
- with sand, the cell membrane broke -> no individual cells

result: tumor is not caused by another tumor cells. trigger of sarcoma = RSV, a single viral particle

RSV genome

contains core proteins, reverse transcriptase, envelope protein, and Src

first human oncogene

Ras oncogene

Ras oncogene

a constitutively activated form of RAS (KRAS, HRAS, NRAS). can stimulate cell growth without any growth factor
- proto-oncogene = ras, oncogene = mutated ras

how proto-oncogenes beome oncogenes

1) mutation (point/delete/inser) -> hyperactive protein

2) gene amplification -> greatly overproduced

3) chromosome translocation (promoter/gene fusion) -> abnormal production of normal protein or novel protein

classification of oncogenes

1) growth factors (PDGF)
2) growth factor receptors (EGFR, PDGFR)
3) signal transducers (Ras, Src, Abl)
4) cell cycle regulators (cyclin D, CDK4)
5) transcription factors (Myc, MITF)
6) anti-apoptotic regulators

BCL-2

oncogene; translocations lead to over-expression -> favors prolonged survival of the cell and confers resistance to the cytotoxic effect of chemotherapeutic agents

MYC expression

transcription factor oncogene
patients: > 10 copies of N-myc = lower rate of survival

mice: bcl-2 alone = lived
myc alone = less likely to live
myc + bcl-2 = guaranteed death

Philadelphia chromosome

ABL translocation from normal chromosome 9 onto chromosome 22
- found in 90% of chronic myeloid leukemia (CML)
- gives rise to a BCR-ABL fusion protein (p210) with constitutive tyrosine kinase activity
- inhibited by Gleevec

Gleevec/imatinib

competitive inhibitor of BCR-ABL

blocks ATP binding to enzyme -> block chromosome formation -> block downstream signaling cascade

tumor suppressor genes

encoded proteins that negatively regulate cell proliferation in their normal state
- lost or inactivated in many tumors, contributing to abnormal proliferation of tumor cells (P53, PTEN, etc)

Rb

cell cycle regulator; first human tumor suppressor gene ID'd
- retinoblastoma requires loss of both functional copies of Rb gene

p53

inactivated in many cancers; mutations of p53 in about 50% of all cancers
- normally responsible for cell cycle arrest and apoptosis

PTEN

lipid a protein phosphatase mutated in many cancers

oncogenes vs tumor suppressor genes

- oncogenes: activation increases cancer risk. mutated form of proto-onco gene. gain-of-function mutation can overactivate a proto-oncogene to make it an oncogene

- tumor suppressor genes (TSG): activation decreases cancer risk; loss-of-funcion can lead to loss of activity, allowing for cancer to occur

resisting cell death

avoiding assisted suicide of outlaw cells; abrogation of the inborn willingness of cells to die for the benefit of the organism
- increases expression of anti-apoptotic Bcl-2 family proteins
- down regulating pro-apoptotic Bcl-2 family members

Bcl-2 and B cell lymphoma

alterations in the Bcl-2 family of proteins (increased expression -> anti-apoptosis)

enabling replicative immorality

circumventing a counting mechanism that disrupts continuing cell division when a set limit is reached
- telomere activity related

telomere

a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes

telomeres and cancer

- cancer cells maintain their telomeres (90% by increasing production of telomerase)
- telomerase: adds telomeric DNA to the ends of chromosomes

hTERT

telomerase reverse transcriptise
- in HEK cells: without hTERT, eventually die. with hTERT, HEK cells keep growing

inducing angiogenesis

turning on new blood vessel growth to feed and nurture the growing mass of cancer cells
- hypoxia-inducible transcription factor (HIF) system

hypoxia-inducible transcription factor (HIF) system

genes that indirectly/directly induce angiogenesis and other stress-adaptive capabilities of cancer cells

1) tumor secretes VEGF
2) VEGF increases blood vessel expression and movement to tumor
3) tumor has increased blood supply

activating invasion and metastasis

- malignant tumor cells tend to invade locally and metastasize to other organs
- EMT and seed and soil hypothesis

epithelia mesenchymal transition (EMT)

epithelia cells acquire mesenchymal traits
- loss of adherent junctions, change in cellular morphology, increased motility
- roughly 90% of cancers arise as carcinomas in epithelial (surface) tissue

tumor cell invasion and metastasis

tumor splits into mesenchymal-like cancer cells of a collective invasion of epithelial-like + mesenchymal-like cancer cells -> intravasation -> metastasis -> extravasation

seed and soil hypothesis

tumor cells grow preferentially in the microenvironment of select organs
- metastases resulted only when the appropriate seed was implanted in its suitable soil
- 50% related to brain; might be because brain is a hospitable environment