CELLULAR SIGNAL TRANSDUCTION 2) From the membrane to the nucleus

Part F

TRANSCRIPTION FACTORS AND THE

REGULATION OF TRANSCRIPTION

Rainer deMartin / Erhard Hofer Institute of Vascular Biology and Thrombosis Research

Vienna Competence Center Lazarettgasse 19, 1090 Wien

Rainer deMartin: Principals of transcriptional regulation Structural features of transcription factors Basic mechanisms of transcriptional regulation Erhard Hofer Activation of transcription factors by surface receptors: Summary selected examples discussed in part B: CREB, SRF, NFAT, SMAD Some additional specific example: Regulation of signaling pathways/transcription factors by proteolytic cleavage: WNT, NOTCH (embryonic development, adult stem cells) Nuclear import of transcription factors Chromatin and transcription Transcription initiation complexes

SMAD

SMAD/Co-SMAD

SMAD

STAT STAT A-Cyclase PLC Ras

STAT

cAMP

Signaling pathways: Receptor to transcription factors

PKA

SRF

IKKK

NFAT / NFκB

CREB, SRF, NFAT, SMAD

Gene regulation by PKA:

CREB bound to CRE is phosphorylated by PKA, activates transcription without effect on DNA binding

Example 3

Gene regulation via MAPK pathway (activated by Ras or PLC-γ/PKC): The phosphorylated MAPK ERK is transported into the nucleus and phosphorylates the transcription factor TCF on the promoter

ERK: extracellular signal regulated kinase TCF: ternary complex factor SRF: serum response factor SRE: serum response element (DNA binding sequence for TCF and SRF in promoter of various genes)

Genes for Cell cycle/ Proliferation early response genes, e.g. c-fos

or: PLC-γ Raf MEK

Ca++

Calmodulin

Calcineurin

NFAT

P I

Gene regulation via Ca++ Signaling pathway - the Phosphatase Calcineurin dephosphorylates NFAT NFAT translocates into nucleus NFAT= transcription factor (nuclear factor activated T cell) Kern

P

Gene regulation via TGF-β-R / SMADs: Transport of phosphorylated SMADs into nucleus

Regulation of transcription factors

by proteolytic cleavage

WNT signaling pathway Secreted signaling peptide, important in embryonic development, mutatios on proteins of wnt signaling pathway frequent in cancer

Example 4a

Wnt Wingless (Drosophila) Int-1 (breast cancer oncogen) (detected experimentally by virus Integration) In signalling pathway: APC (adenomoteous polyposis coli protein) mutated in adenoma of colon and 80 % of colon cancer induces myc gene and proliferation

(LDL rceptor related protein)

(Signaling protein)

(Corepressor)

β-Catenin signaling pathway: w/o signal: β-catenin is continously phosphorylated, ubiquitinylated, degraded in proteasom Wnt-signal: Kinase is inhibited, non-phosphorylated β-Catenin transported into nucleus, activates transcription by competing with a corepressor

(β-Catenin Coaktivator)

Phosphorylation, Ubiquitinylation, degradation in proteasom

components of the WNT signaling pathways e.g. important for maintaining the stem cell population in gastro-intestinal tract, over-activation by APC mutation – leads to colorectal cancer

Notch hedgehog

1- Embryonic development e.g. nerve cells Drosophila, Delta on nerve cells signals to neighbouring cell: do not differentiate to nerve cell, peptide translocates into nucleus and converts CSL to become an activator 2- Angiogenesis: Tip versus stalk cell, tip cell signals stalk cell not to become another tip cell

Example 4b

Nuclear import / -export

Nuclear membrane: Double membrane with nuclear pores

26 nm

Nuclear pore complex

innere Seite

The nuclear localization signal is a basic amino acid sequence

Model of nuclear import mediated by the small G-protein Ran

Model of nuclear import and export

CHROMATIN

Summary of chromatin structure

Interphase

Metaphase

Eigenschaften von Insulatorelementen

model

Change of chromatin structure by activator protein

Ac

Ac

HAT

HDAC

condensed CHROMATIN no TRANScRIPTION Z.B. HETEROCHROMATIN

loose CHROMATIN TRANCRIPTION Z.B. EUCHROMATIN

HAT = HISTONACETYLTRANSFERASE ACTIVATOR OF TRANSCRIPTION HDAC = HISTONDEACETYLASE REPRESSOR of TRANSCRIPTION

DNA

NUCLEOSOM

CHROMATIN

GENEXCS18

A

REPRESSION

ACTIVIERUNG

HDAC

X

Y

HAT

Target GENE

Target GENE

Z

REGULATION OF TRANSCRIPTION BY CHANGE OF CHROMATIN STRUCTUR

condensed Chromatin

open Chromatin

Repressor

Aktivator

GENEXCS19

Gene regulatory proteins form frequently large

complexes

DNA Looping

Mediator

Gene expression