Action at a Distance Bacterial and eukaryotic enhancers stimulate transcription even though located some distance from their promoters Four hypotheses.

Name: Action at a Distance Bacterial and eukaryotic enhancers stimulate transcription even though located some distance from their promoters Four hypotheses.
Uploaded: 2017-08-19T06:36:14+00:00
Duration: PTM30S4
Channel: Drusilla Copeland
Description: Action at a Distance Bacterial and eukaryotic enhancers stimulate transcription even though located some distance from their promoters Four hypotheses.

Action at a Distance Bacterial and eukaryotic enhancers stimulate transcription even though located some distance from their promoters Four hypotheses attempt to explain the ability of enhancers to act at a distance Change in topology ie. supercoiling Sliding Looping Facilitated tracking

Hypotheses of Enhancer Action
Change in topology Sliding Looping Facilitated tracking

Complex Enhancers Many genes can have more than one activator-binding site permitting them to respond to multiple stimuli Each of the activators that bind at these sites must be able to interact with the preinitiation complex assembling at the promoter, likely by looping out any intervening DNA

Control Region of the Metallothionine Gene
The metallothionine gene product helps eukaryotes cope with heavy metal poisoning Turned on by several different agents Complex enhancers enable a gene to respond differently to different combinations of activators This gives cells exquisitely fine control over their genes in different tissues, or at different times in a developing organism

After identifying individual regions in the 5’ UTR of the Endo16 gene that bind nuclear proteins, the isolated binding regions were fused to a reporter cassette and reintroduced into sea urchin. Expression was monitored and it was determined that some regions act alone and others in combination with each other. Very important for the fine control of gene expression required during development..

Architectural Transcription Factors
Architectural transcription factors are those transcription factors whose sole or main purpose seems to be to change the shape of a DNA control region so that other proteins can interact successfully to stimulate transcription. Important when control regions are in very close proximity to one another.

Short DNA acts as rigid rod
Long DNA behaves more like a string and can be easily manipulate

Example of Architectural Transcription Factor: Control region of the human T-cell receptor alpha chain (TCR ) gene Within 112 bp upstream of the start of transcription are 3 enhancer elements These elements bind to: Ets-1, LEF-1, CREB LEF-1 alone cannot activate gene. Role? bends DNA at the minor grove by 130deg

Used synthetic DNA containing LEF-1 binding site in electrophoretic assay to show DNA bending
When the site was positioned in the middle of the synthetic DNA, movement through a gel was greatly retarded

DNA Bending Aids Protein Binding
The activator LEF-1 binds to the minor groove of its DNA target through its HMG domain and induces strong bending of DNA LEF-1 does not enhance transcription by itself Bending it helps other activators bind and interact with activators and general transcription factors

Enhanceosome An enhanceosome is a nucleoprotein complex containing a collection of activators bound to an enhancer in such a way that stimulates transcription Ex. IFN-beta contains 8 binding sites which must all be occupied by activators. The other end of the simple/complex enhancer spectrum only activated when a cell is under attack by a virus.

Dilema: Some enhancers act at great distances from their promoters. ie
Dilema: Some enhancers act at great distances from their promoters. ie. Drosophila cut locus is 85kb from promoters. Enhancer is likely to come in proximity to other genes, how does the cell prevent inappropriate activation?

Insulators Insulators can shield genes from activation by enhancers (enhancer blocking activity) Insulators can shield genes from repression by silencers (barrier activity). Prevent condensing Insulators define regions between DNA domains. ie. Between enhancers and promoters

Mechanism of Insulator Activity
One mechanism which can be ruled out is that insulators induce the condensation of DNA upstream of their location. If a gene were placed upstream of such an insulator, it would always be silenced Experiments in Drosophila show that such genes can still be active and can be activated by their own enhancers.

Mechanism of Insulator Activity
Sliding model Activator bound to an enhancer and stimulator slides along DNA from enhancer to promoter Looping model Two insulators flank an enhancer, when bound they interact with each other isolating enhancer

However…some insulators work as single copies.
ie. The Drosophila hairy-wing insulator - single insulator = some insulator activity - two insulators = no insulator activity - insulator-enhancer-insulator = increased insulator activity

Model of Multiple Insulator Action
Canceling of insulator activity

Summary Some insulators have both enhancer-blocking and barrier activities, but some have only one or the other Insulators may do their job by working in pairs that bind proteins that can interact to form DNA loops that would isolate enhancers and silencers so they can no longer stimulate or repress promoters Insulators may establish boundaries between DNA regions in a chromosome

12.6 Regulation of Transcription Factors
Several activators do not active transcription by contacting the basal transcription apparatus directly. Rely on: Coactivators = no activator function on its own, but collaborates with one or more activators to stimulate the expression of a set of genes

Coactivator Activator

Phosphorylation of activators can allow them to interact with coactivators that in turn stimulate transcription

Model for the Activation of a Nuclear Receptor-Activated Gene

Sometimes genes are inactivated by the destruction of their activators Ubiquitylation of transcription factors can mark them for Destruction by proteolysis Stimulation of activity Sumoylation is the attachment of the polypeptide SUMO which can target for incorporation into compartments of the nucleus Methylation and acetylation can modulate activity

Ubiquitylation Normal function of ubiquitylation is to mark proteins for destruction by the proteasome. ie. Aprx 20% of all proteins are made incorrectly and need to be quickly disposed of A fine balance may exist between coactivators and corepressors which have ubiquitylating ability - ie. A corepressor may mark a coactivator for destruction, tipping the scale towards repression

Activator Sumoylation (SUMO or small ubiquitin like modifier)
Sumoylation is the addition of one or more copies of the 101-amino acid polypeptide SUMO (Small Ubiquitin-Related Modifier) to lysine residues on a protein Process is similar to ubiquitylation Results quite different – sumoylated activators are targeted to a specific nuclear compartment that keeps them stable

Activator Acetylation
Nonhistone activators and repressors can be acetylated by HATs HAT is the enzyme histone acetyltransferase which can act on nonhistone activators and repressors Such acetylation can have either positive or negative effects - ex. p53 acetylation by coactivator p300 results in increased DNA binding

Signal Transduction Pathways
Signal transduction pathways begin with a signaling molecule interacting with a receptor on the cell surface This interaction sends the signal into the cell and frequently leads to altered gene expression Many signal transduction pathways rely on protein phosphorylation to pass the signal from one protein to another This leads to signal amplification at each step

Three pathways that use CBP/p300 to mediate transcription activation

Ras and Raf Signal Transduction

Lecture key words Cell cycle Transcription factors Phosphorylation
Heterodimerization Immunohistochemistry Immunprecipitation Growth factors Apoptosis Colony formation Nuclear localization Consensus sequences Motifs

CDCA7 | a case study in cellular regulation
Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis  CANCER

Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis  CANCER Modes: -phosphorylation, -subcellular localization - heterodimerization

CDCA7 | What is known Myc and E2F target gene with peak expression at G1-S Novel member of cell division cycle-associated gene family Frequently overexpressed in human tumors JPO2 binds Myc and promotes Myc dependent transformation JPO2 and CDCA7 share cysteine rich C-term which may bind DNA Not known if CDCA7 interacts with Myc

Myc | Just the facts Discovered in Burkitt’s lymphoma patients
Member of bHLH-LZ family of transcription factors Requires heterodimerization with Max to transactivate Regulates the expression of ~10-15% of genes Role in development, cell division, cell growth, metabolism, angiogenesis Early response gene induced by growth factors, levels peak at G0-G1 Driving force of cell cycle and malignant transformation Active in 70% of human cancers ~100,000 cancer deaths per year in the US due to changes in Myc

TOR rictor Growth Factors PI3K PIP2 PIP3 PDK1 Receptor Tyrosine Kinase
14-3-3 P P Cytoplasm AKT P 14-3-3 AKT P CDCA7 P P 14-3-3 rictor TOR 14-3-3 P CDCA7 P AKT 14-3-3 14-3-3 P CDCA7 Myc Myc Transcription Pro-apoptotic Genes ? Nucleus

Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis  CANCER Modes: -phosphorylation -subcellular localization -heterodimerization

CDCA7 | Conservation AKT consensus site R X R X X T/S F/L
CDCA7 T R P R R R T F >90% conserved human monkey dog mouse chicken frog zebrafish 24 49 69 78 112 190 261 363 AKT kinase 0.005% 1 T163 371 humCDCA7 zinc finger 261 361

CDCA7 is phosphorylated at t163
Many ways to prove phosphorylation Custom made antibody against phospho-T163 Radioactivity and mutational analysis Phosphotase CDCA7 CDCA7+ CIP T163A T163A + CIP Vector a-FLAG a-P-T163 WT T163A

CDCA7 is phosphorylated at t163
a-FLAG a-P-T163 Vector 5 15 45 120 360 PDGF (min) Merge Ratio P-T163/ Total CDCA7 1.0 2.2 3.6 4.7 4.0 3.9 Treatments w/ growth factors T= 0’ T= 20’ T= 30’ Immunohistochemistry T= 40’ T= 50’ T= 60’

CDCA7 is phosphorylated at t163 by AKT
Inhibitors Vector CDCA7 Akt inh VIII IP: a-FLAG Blot: a-P-T163 Blot: a-FLAG

Where is cdca7 found? T163A CDCA7 a-Flag DAPI

CDCA7 | Conservation How do we test for a nuclear localization signal?
>90% conserved human monkey dog mouse chicken frog zebrafish 24 49 69 78 112 190 261 363 1 T163 371 humCDCA7 NLS? NLS? zinc finger 261 361 RRPRRRTFPGVASRRNPERRARPLTRSRSR How do we test for a nuclear localization signal? Isolate region in question and test its ability to target an innocuous protein to the nucleus

CDCA7 contains an NLS Passive diffusion into nucleus <45 KDa
SV40 SV40 KE R171E CDCA7 CDCA7 R176E R171/176E CDCA7 (T163A) R176/184E R184E 157-RRPRRRTFPGVASRRNPERRARPLTRSRSRIL-188 Supplementary Figure 2

Phosphorylation alters localization
a-CDCA7 Nuclei Merge Unstimulated PDGF PDGF + LY Supplementary Figure 3

CDCA7 | Conservation 157-186 RRPRRRTFPGVASRRNPERRARPLTRSRSR
>90% conserved human monkey dog mouse chicken frog zebrafish 24 49 69 78 112 190 261 363 1 T163 371 humCDCA7 NLS? NLS? zinc finger 261 361 RRPRRRTFPGVASRRNPERRARPLTRSRSR consensus binding site R-[S/F/Y]-X-pS/T -X -P cdcA7 T R R R T F P Mekk2 T G R K T F P

| Just the facts Large family of highly conserved, small, acidic polypeptides of kDa Seven different isoforms in humans, σ directly implicated in cancer Binds to protein ligands at defined phospho-serine/threonine motif RSXpS/TXP Over 200 known ligands regulates process relevant to cancer biology: cell-cycle progression, apoptosis and mitogenic signaling

| Modes of influence exists as a dimer and offers two binding sites for phospho-S/T motifs Can function as adaptor protein for: a) two proteins that would otherwise not associate b) one protein with two motifs = high affinity Adapted from Hermeking, 2005 Affects change by: Alteration of enzymatic activity – maintains RAF1 in inactive state Alteration of DNA-binding activity – increases p53 DNA-binding after DNA damage Sequestration - BAD, FKHRL1, HDAC5 and CDC25C are localized to cytoplasm Altering protein-protein interactions - reduced affinity of CDC25A to CDC2 Adaptor protein functions – bridging of RAF1 to BCR Sequestration Altering protein-protein interactions

CDCA7 binds14-3-3 and is phospho dependent
Western blots S/F/Y consensus site R X pT X P Wildtype Vector R158A P159A R160A R161A R162A T163A F164A P165A Blot: a-FLAG a-P-T163 a

14-3-3 alters CDCA7 localization
R161A CDCA7 T163A R161A/ a-Flag DAPI Is masking the NLS within the T163 region?

CDCA7 | What is known Myc and E2F target gene with peak expression at G1-S Novel member of cell division cycle-associated gene family Frequently overexpressed in human tumors JPO2 binds Myc and promotes Myc dependent transformation JPO2 and CDCA7 share cysteine rich C-term which may bind DNA Not known if CDCA7 interacts with Myc

CDCA7 binds the transcription factor Myc
Co-immunoprecipitation His-Myc Pulldown Blot: a-FLAG Input WT CDCA7 T163A CDCA7 D( ) CDCA7 D(1-146) CDCA7 D(1-172) CDCA7 D(1-202) CDCA7 D(1-234) CDCA7 D( ) CDCA7 D( ) CDCA7 D( ) CDCA7 D( ) CDCA7 WT T163A D( ) D(1-146) D(1-172) D(1-202) D(1-234) D( ) D( ) D( ) D( ) + - CDCA7

So how does cdca7 affect phenotype?
Apoptosis proliferation

14-3-3/CDCA7 binding influence Myc-induced transformation
Colony formation assay

14-3-3/CDCA7 binding influence Myc-induced apoptosis
Rat1 Trypan blue exclusion Myc-Rat1 Sh1-Myc-Rat1

TOR rictor Growth Factors PI3K PIP2 PIP3 PDK1 Receptor Tyrosine Kinase
14-3-3 P P Cytoplasm AKT P 14-3-3 AKT P CDCA7 P P 14-3-3 rictor TOR 14-3-3 P CDCA7 P AKT 14-3-3 14-3-3 P CDCA7 Myc Myc Transcription Pro-apoptotic Genes ? Nucleus

summary CDCA7 is a novel target of AKT required for Myc-dependent apoptosis Phosphorylation of T163 inhibits CDCA7/Myc apoptosis by: Promoting binding Disruption of Myc binding Shuttling to the cytoplasm Potential for medical intervention in Myc tumors where AKT is dysregulated

Action at a Distance Bacterial and eukaryotic enhancers stimulate transcription even though located some distance from their promoters Four hypotheses.

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Action at a Distance Bacterial and eukaryotic enhancers stimulate transcription even though located some distance from their promoters Four hypotheses.

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Presentation on theme: "Action at a Distance Bacterial and eukaryotic enhancers stimulate transcription even though located some distance from their promoters Four hypotheses."— Presentation transcript:

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