Molecular and Cell

Biology I Lecture 14

Transcription: Transcription
Regulation I

Sevinc Ercan
se71@nyu.edu
Student hours: Wednesday 11 am – 12 pm
Zoom link from syllabus “information and
policies”
1
Yeast one hybrid – quest for the TF that regulates
zen
DNA sequence of the DNA fragment that activates zen
transcription:

Chris Rushlow
Lab
 Quest has ended with identification of Zelda

DNA sequence of the DNA fragment that activates zen

transcription:

Yeast one hybrid assay

Found a transcription factor and named it Zelda (Zld; Zinc-finger early Drosophila
activator)

Chris Rushlow
Lab
How do we test that Zelda binds to these specific
motifs?

DNase I footprinting
assay

Melissa Harisson
Lab
DNase I footprinting

TF
200 bp

Melissa n
Harisso
 Zelda
DNase I footprinting mixed with DNA containing wild type (WT) or
mutated (MUT) motif
I can
1 2 conclude from this DNase I footprinting result that
3 Zelda binds to its motif specifically* What lane best
4 support this conclusion?*meaning it binds to this
particular DNA sequence motif

Melissa Harisson 7
Lab
8
Does Zld bind to the different motifs similarly?

~20-40
bp DNA
Gel-shift
assay
T
F

Chris Rushlow 9
Lab
Gel shift assay

Amount of
Cont TF in the
rol
prot tube
ein

1
3 4
2
 Question: Gel shift assay
“Zld binding to oligonucleotides containing different TAGteam sites (denoted beneath each
section of the gel). The first lane in each section contains free probe, the second lane contains
probe plus 10 ng GST–ZldC, and the third lane contains probe plus 30 ng GST–ZldC.”
How do you know that Zelda binds
specifically to the motif?
What is the difference between Zelda
binding to the three motif sequence versions tested?
How do the DNA sequence of
three motifs differ?

muta Chris Rushlow 1

motif motif motif Lab 2
1 2 3 ted
1
3
Question: Gel shift assay

“Zld binding to oligonucleotides containing different TAGteam sites (denoted beneath each
section of the gel). The first lane in each section contains free probe, the second lane
contains probe plus 10 ng GST–ZldC, and the third lane contains probe plus 30 ng GST–
ZldC.”

How do the DNA sequence

of three motifs differ?

motif motif motif muta

1 2 3 ted
moti
f Chris Rushlow 1
Lab 4
Question: Gel shift versus DNase I footprinting

Imagine you identified a 200 bp region that controls activation of a gene.

You used the 200 bp fragment in yeast one hybrid and identified that TF1 binds to
it. Despite various attempts, you were not able to narrow down to a sequence
motif.
Which assay would you use to determine the DNA sequence motif that
TF1 recognizes?
Why?
Transcription regulation in eukaryotes

Activation domain
DNA binding
domain

In prokaryotes we learnt small molecules regulating TF binding.

Examples?
How are eukaryotic TFs’ binding regulated?
- Small molecules e.g. hormone receptors
- Combinatorial binding of TFs
- DNA accessibility
Hormone receptors are TFs that bind to lipid soluble hormones

Fig 8-42 9th

ed
General design of transcription factors in the nuclear-receptor superfamily

Contains a Contains
repeat of a
Variable the C4 zinc- hormone
regions finger -
function as motif depende
activation nt
domain(s) in activatio
most nuclear n
receptors domain
 Consensus sequences of DNA response elements that bind 5 nuclear receptors

glucocortico
id
estrogen

retinoid X receptor H
(RXR) o
m vitamin
RXR + o D3
VDR d
i
m thyroid
RXR+ hormone
TR e
r
s
RXR+R retinoic
H acid
AR e
t
e How does the hormone regulate the
r receptor?
o 1) localization
d 2) activation domain function
Fig 8-44 9th i
ed
m
e
GR binding regulates its translocation to the nucleus

Fig 8-45 9th

ed
 What domain of GR is sufficient for hormone-induced nuclear translocation?

Glu
cort Fig 8-45 9th
ed
icoi
d Poll: If the ligand binding domain is sufficient for hormone induced
hor nuclear translocation, would you expect a result as in (a) or (b)?
mo A – left hand up B – right hand up
ne
dex
am
 What domain of GR is sufficient for hormone-induced nuclear translocation?

Glu
cort Fig 8-45 9th
ed
icoi
d
hor How does the hormone regulate the receptor?
mo 1) localization
ne 2) activation domain function
dex
am
 Ligand binding to estrogen receptor changes its interaction with co-activator

Fig 8-26 9th

ed

agonist=a substance that fully activates the receptor that it attaches

antagonist=a substance that attaches to a receptor but does not activate it or kicks out
agonist
The green alpha helix interacts with the ligand (estrogen).
Ligand binding domain (dark helices) interact with co activator
. The inactive conformation of the estrogen receptor is stabilized by 2
3
tamoxifen.
Transcription regulation in eukaryotes

In prokaryotes we learnt small molecules regulating TF binding.

Examples?
How are eukaryotic TFs’ binding regulated?
- Small molecules e.g. hormone receptors
- Combinatorial binding of TFs
- DNA accessibility
Combinatorial binding allows tight control of transcription

Interleukin
-2 (IL2)

IL2 is a signaling molecule that controls immune response. Abnormal expression

can cause autoimmune diseases such as rheumatoid arthritis.
 Combinatorial binding allows tight control of transcription
AP-1 is a heterodimer of c-Jun and c-Fos.
Activated by growth factors & other
NFAT is activated signals.
by calcium
signaling.

Interleukin
-2 (IL2)

IL2 is a signaling molecule that controls immune response. Abnormal expression

can cause autoimmune diseases such as rheumatoid arthritis.
Combinatorial regulation is a mechanism that enables tight control of gene expression,
via integration of multiple signaling pathways that control different transcription
factors.
 Combinatorial regulation increases gene-control options

Gene Gene Gene Gene Gene Gene

1 2 3 4 5 6
Tissue
1(A):
Tissue
2(B):
Tissue
3(C):
Tissue
4(AB): Fig 8-27 9th
Tissue ed ~1400 transcription factors control ~21,000 human
5(AC): genes
Tissue
6(BC):