Gene Expression

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Epigenetics: DNA Methylation
Genomic Imprinting

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- for majority of genes, an allele from either the father or the mother may be silenced so that only the one remaining allele will be expressed
- CG islands: CG dinucleotides are found mostly in
gene promoter regions; when cytosines become methylated at C5, gene expression is "silenced"

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DNA methylases (DNMT)

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- de novo methylation: In newly developing zygotes, CG islands of specific genes are recognized by "DNA methylases (DNMT)" and cytosine is methylated on both strands; scaffolding proteins bind such mCG regions, promoting DNA condensation, and ultimately silencing gene expression altogether
- maintenance methylation: During normal replication, DNMTs recognize mCG in the parental strand, and methylate cytosine in the corresponding complementary GC pair in the daughter strand; inactivates one chromosome
- cancer: DNMTs are known to silence expression of "tumor suppressor" genes

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Epigenetics: DNA Methylation
X-Chromosome Inactivation

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- Process by which one of the two
X chromosomes in female mammals is packaged
into heterochromatin, thus silencing transcription
of nearly all its genes= Xi
- Xi has high levels of DNA (CG) methylation and low levels of histone acetylation compared to Xa
- Xi can viewed as a discreet dark
staining body in the periphery of the nucleus= Barr bodies

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Calico

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- results from random X-inactivation in melanocytes that grow during embryonic development

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Structure of Nuclear Receptors

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- ligand binding domain (LBD)- binds to a specific
ligand, which stimulates its transcriptional activation function (AF2); C-terminus
- DNA binding domain (DBD)- transcription factor that binds specific regions of DNA called hormone response elements (HRE)
- N-terminal regulatory domain (NRD)- contains the
"transcriptional activation function (AF1), which
together, with AF2 bind coregulators for chromatin remodeling
-hinge: A flexible region between DBD and LBD

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Type I Nuclear Receptors

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- binds receptor in cytoplasm causing:
- release of repressor proteins
- receptor homo-dimerization
- receptor translocation to nucleus
- receptor binding to DNA response element
- and recruitment of coregulators
- receptors for estrogen, androgen, progesterone, and glucocorticoid

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Type II Nuclear Receptors

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- binds receptor in nuclues causing:
- release of corepressor proteins
- hetero-dimerization (requiring two different
signaling molecules to elicit a transcriptional response)
- DNA binding
- and recruitment of coactivator proteins
- receptorsvarious forms of vitamin A, vitamin D, and thyroid hormones

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Regulation of mRNA Transcription:
Nuclear Receptors

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- number of steroid hormones, vitamins, and assorted other "ligands" diffuse into cells and specifically bind their target receptor-> conformational change
- enhances DNA binding to "hormone response elements" and leads to recruitment and activation of transcriptional "coregulators"

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Coregulators

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- coregulators are histone modifying enzymes
a) "Coactivators" cause release of histones from DNA (e.g., HATs); ex. acetylation
b) "Corepressors" cause condensation of nucleosomes (e.g., HDATs)

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Regulation of mRNA Transcription:
G-Protein Coupled Receptors

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- cell surface protein receptors with seven α-helices that span the lipid bilayer in alternating fashion
- Residues in "outer loops" provide specific binding to a large variety of small molecules, protein hormones and neurotransmitters
- Residues in "inner loops" respond as GEFs, activating exchange of bound GDP for GTP in the associated "heterotrimeric G-protein"
- cAMP and Wnt Canonical Pathway

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Heterotrimeric G-protein

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- 3 subunits but only larger α-subunit binds GTP/GDP and then associates with β- and γ-subunits, which together are associated with inner loops of GPCRs
-When bound to GTP, the α-subunit dissociates from the GPCR/βγ complex and associates and activates the transmembrane (TM) enzyme, adenylyl cyclase

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Adenylyl Cyclase (AC)

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Contains six TM α-helices, followed by a cytoplasmic "C1 domain", another six TM α-helices, and the final cytoplasmic "C2 domain"
- C1/C2 domains associate to form the active site and catalyze production of "3',5'-cyclic AMP (cAMP)" and PPi from ATP
- cAMP is a type of second messenger (outside signal caused production of inside signal that binds to protein kinase)

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cAMP-dependent protein kinase (PKA)

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- tetramer made up of dimer of regulatory subunits (R1/R2) which are associated with and inhibit dimer of catalytic subunits
- "cAMP" binds the R subunits, which causes them
to release the active PKA subunits

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cAMP response element binding protein
(CREB)

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- Activated PKA translocates to the nucleus and phosphorylates CREB (transcription factor), which causes it to dimerize and bind the "cAMP response element (CRE)" which then recruit/activate various transcriptional coregulators (some of which are histone modifying proteins that set of transcription)
- DNA binding domain is a basic leucine zipper (bZIP)
- regulates expression of numerous and different genes in different organs; learning and memory

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Phosphodiesterase (PDE)

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- Activated PKA that remains in the cytoplasm phosphorylates PDE, which activates its ability to hydrolyze cAMP to AMP
- cAMP comes off of R subunits and thus they can bind and sequester active PKA subunits, attenuating the downstream signaling events

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cAMP Pathway Summary

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- extracellular signal binds to G-protein coupled receptor eventually turns it into GEF→ α-subunit of G protein releases GDP and takes up GTP→ α-subunit released from receptor and associates with transmembrane enzyme AC→ catalyzes cyclization reaction of ATP to form cAMP
- cAMP binds PKA releasing active form that goes to nucleus→ catalyzes phosphorylation of CREB which dimerizes and binds CRE→ recruitment of coregulators including histone modifying proteins→ transcription
- attenuated by PDE

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PKA

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- also recognized for its role in controlling
enzyme activities related to stress responses
a) Starvation: Low blood sugar is counteracted by "glucagon" action on cAMP
b) Fight-or Flight: Rapid increased heart rate and respiration, with production of needed glucose fuel is stimulated by catecholamine action on cAMP production
- Catecholamines: "epinephrine, "norepinephrine", and "dopamine"

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Wingless-related integration site (Wnt)

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- family of highly palmitoylated glycoproteins that cells secrete to initiate a developmental response

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Frizzled (Fz)

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- Family of GPCRs that serve as cell surface receptors for Wnt signaling
- Cys-rich (5 disulfide) extracellular region binds Wnt, in cooperation with its coreceptor, the lipoprotein receptor related protein (LRP)

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Dishevelled (Dsh)

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- multidomain adaptor protein that associates with cytoplasmic loops of Wnt-bound Fz receptor
- then binds and inhibits the multi-protein destruction complex

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Destruction complex (GSK3)

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- numerous proteins and enzymes that sequester the transcriptional regulator, β-catenin, and target it for degradation
- In the complex, β-catenin is phosphorylated by
“glycogen synthase kinase-3 (GSK3)”, which signals it for ubiquitination
- β-catenin (β-Cat)- cytoskeletal protein but also a moonlighter (same gene product acting with different functions) with significant role in transcriptional regulation

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Wnt Canonical Pathway Summary

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- Wnt signal is bound by Fz→ Fz is activated→ DSH associates with Wnt- bound Fz receptor→ Dsh recruits and sequesters destruction complex→ β-Cat is released→ goes to nucleus and acts as transcription factor
- cutting edge cancer biology

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Regulation of mRNA Transcription:
Enzyme/ Kinase- Linked Receptors

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1. JAK-STAT Pathway
2. MAPK Pathway

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Cytokine

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- Extracellular signaling proteins that are secreted by cells, primarily for developmental and immunological responses

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Cytokine receptor (CR)

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- family of cell surface receptors, composed of an (i) “extracellular domain”, (ii) a “transmembrane domain”, and a (iii) “cytoplasmic domain”
- binding any number of particular extracellular "cytokines" causes CR monomers to associate into dimers, and likely higher order structures
- this results in Tyr phosphorylation of their cytoplasmic domains by "Janus kinases (Jak)"

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Janus kinase (Jak)

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- family of intracellular "nonreceptor tyrosine kinases" that are associated and activated by cytokine-bound receptors
- phosphorylate Tyr residues in the cytoplasmic
domain of bound receptors
- "Stat" family of transcription factors bind to pTyr using their central "SH2 domain", which is next phosphorylated by Jak

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Signal transducer & activator
of transcription
(Stat)

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- Family of transcription factors that have 3 domains: DNA binding, phosphate binding (SH2), and transactivation domains
- recruited to membrane where binds pTyr in activated cytokine receptors
- also become phosphorylated by Jaks causing dimerization (homo-/hetero-), nuclear transport, and DNA binding

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JAK-STAT Pathway Summary

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- cytokines bind to extracellular domain of cytokine receptor→ Janus kinase associates with receptor→ kinase then phosphorylates Tyr of receptor's cytoplasmic domains→ STAT binds to phosphorylated Tyr by SH2 domain→ STAT monomers phosphorylated by Jaks and then dimerize→ transported to nucleus in form of beta- lucine zippers= basic on one side so bind DNA

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Receptor tyrosine kinase (RTK)

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- family of cell surface receptors, composed of an (i) “extracellular domain”, (ii) a “transmembrane domain”, and a (iii) “cytoplasmic tyrosine kinase domain” which acts as receptor
- binding of any number of particular extracellular
“growth factors, cytokines, or hormones” causes RTK
monomers to associate into dimers, and likely higher order structures
- results in the cytoplasmic kinase domains catalyzing phopshoylation of Tyr residues in the C-terminal tail region
- serve to recruit and assemble signaling complexes

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Growth factor receptor-bound protein-2 (Grb2)

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- Known as an “adaptor protein”, which in general,
contains protein recognition modules that bind and
link enzymes in a cell signaling pathway and do NOT possess enzyme activity
a) one “SH2 domain” specifically binds pTyr in the C-terminus of RTK
b) two “SH3 domains” specifically bind Pro-rich
regions, in this case a downstream effector, Sos (GEF)

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Son of sevenless (Sos)

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- group of “guanine nucleotide exchange factors (GEF)”, which specifically act on the “Ras” subfamily
of “small GTPases”

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Rat sarcoma (Ras)

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- single subunit G-protein, similar in function to the Gα subunit of heterotrimeric G-proteins
- retained at the membrane by any number of types of lipidation (e.g., farnesyl-, prenyl-, or palmitoylation)
- Sos-activated GDP→ GTP results in its binding and activation of Raf, the first protein kinase in the “MAP kinase cascade”
- first oncogene discovered and most prevalent

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Mitogen-activated protein kinases (MAPK)

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- cascading series of protein kinases (e.g., Raf→Mek→Erk), which eventually lead to Erk-catalyzed phosphorylation and activation of numerous transcription factors that drive cell cycle
progression
- Ras-GTP binds Raf, activating its phosphorylation and activation of Mek, which phosphorylates and activates Erk

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Activator protein-1 (AP1)

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-Heterodimeric (Jun-Fos), bZIP transcription factor that regulates gene expression in response to MAPK activation
- Erk phosphorylation of Fos stabilizes chromatin interactions

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MAPK Pathway Summary

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- RTK bind particular extracellular “growth factors, cytokines, or hormones”→ monomers associate into dimers→ cytoplasmic kinase domains catalyze phopshoylation of Tyr residues in the C-terminal tail region→ Grb2 has one SH2 domain that specifically binds pTyr while two SH3 domain specifically the downstream effector, Sos (GEF)→ Sos binds and activates Raf losing GDP and bind GTP→ recruits and binds series of 3 MAP kinases that subsequently phosphorylate one another→ phosphorylation and activation of numerous transcription factors including Fos→

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MAPK Pathway Summary Continued

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associates in heterodimer with Jun to form AP1
- activates transcription of some cell cycle progression proteins to get cell out of G1 phase
- mRNAs exiting nucleus are so potent that they cannot be translated by ribosomes unless coupled so no turning back

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Oncogene

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- normal gene that becomes a cancer causing agent due to hyperactivation by either (i) mutation or (ii) over-expression
- majority are enzymes in cell signaling pathways
that drive cell growth, proliferation, and differentiation
- would qualify as potential "targets" for new "anti-cancer therapies"
- Examples: receptors, kinases, G-proteins, and
transcription factors

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Tumor suppressor (anti-oncogene)

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- normal gene that contributes to cancer progression due to inactivation by either (i) mutation, (ii) deletion, or (iii) silencing
- majority are enzymes in cell signaling pathways that restrain cell growth, proliferation, and differentiation (down regulating, restrain cell growth)
- components are already inactivated, they most
often DO NOT qualify as potential "targets" for
new "anti-cancer therapies"
-Examples: phosphatases, GAPs, and transcription factors

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Know

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- extracellular signals bind and stimulate cascade to regulate both transcription and translation
- know that translation is up-regulated by mTOR

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Stress Responses

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- turn translation off by phosphorylating G protein preventing exchange of GDP for GTP
- kinases that respond to stress by phosphorylating either initiation factor (eIF2) or elongation factor (eEF2)