Regulation of Gene Expression

Aurnab Ghose; 150211

Same genome but the complexity and diversity of the resulting phenotype
is challenging

The dramatic consequences of

gene regulation in biology
Same genome
Different tissues
Different physiology
Different proteome
Different expression pattern

Anise swallowtail, Papilio zelicaon

C-value paradox

C-value paradox
Genome size does not scale with
complexity of the organism
Number of protein-coding genes does not
scale with complexity of the organism
Due to complex ways of regulating genes

The complexity of eukaryotic gene expression regulation

Gene regulatory networks (GRNs)

GRNs are the on-off switches and rheostats of a cell operating at
the gene level. They dynamically regulate the level of expression
for each gene by controlling whether and how vigorously that gene
will be transcribed.
A simple GRN would consist of:
one or more input signaling pathways
regulatory proteins that integrate the input signals
several target genes (in bacteria a target operon)
RNA and proteins produced from those target genes
dynamic feedback loops for regulation of network
architecture and output.

Transcriptional Networks

Transcription factor activities

form the internal representation
of the environmental states

Wyrick and Young, 2002

An environmental sensing mechanism

Signal 1

Signal 2

Signal 3

Signal 4

...

Signal N

Environment

Transcription
factors

X1

X2

gene 2

gene 3

X3

...

Xm

genes
gene 1

gene 4

gene 5

gene 6 ... gene k

Activators increase gene production

X

Activator

No transcription

X binding site

gene Y
Y
Y

Sx
X

Y
Y

X*
X*

Bound activator

INCREASED TRANSCRIPTION

Repressors decrease gene production

Bound repressor
X

Sx
X*
No transcription
X*
Bound repressor
Y

Unbound repressor
X

Y
Y

Transcriptional Networks
Tx factors are themselves encoded by genes regulated by their own Tx factors
Nodes are proteins (or the genes that encode them)
Edges: Tx regulation of one gene by protein products of another (+/- and # s)

20% of transcriptional interactions in E. coli (Shen-Orr, 2002)

Tx networks are dynamical systems:

Input signal
Change in Tx factor activities
Change in protein production rates
Some protein are Tx factors and activate other genes
Other carry out cellular functions
Feedback (e.g. negative feedback autoregulation)

Tx networks have a strong separation of timescales

Input signals changing Tx factor activities: sub-second (~1 ms)
Tx factor binding to DNA equilibrium: seconds (~ 1 s)
Tx & Translation: minutes (~ 5 m)
Accumulation of protein products: several minutes to hours
(50% change in conc. of the translated protein ~ 1h)
(Figures in parentheses for E. coli)

This means the preceding steps can be considered to be at

steady state within the equations that describe the network
dynamics on the slow timescale of changes of the next step.

Tx networks show modularity of their components

One can take DNA from one organism and express it in another
One can choose what promoter to use for this
Promoters and genes are interchangeable
Modularity makes Tx networks plastic during evolution: can
readily incorporate new genes and new regulation
Tx networks evolve rapidly. The edges evolve on a faster
timescale than coding regions

Quantifying the Transcriptome

Measuring gene expression

levels
1. total amount of mRNA = optical density at
appropriate (UV) wavelength
2. mass separation and specific probing, one
gene at a time = Northern blot
3. comprehensive molecular sorting =
microarray technology
1. two-color cDNA or oligo arrays
2. single-color genechips

Northern Blot

cDNA microarray schema

From Duggan et al. Nature Genetics 21, 10 14 (1999)

color code for

relative expression

Quantifying the Proteome

2D-Gel electrophoresis
2-D gel electrophoresis process consists
of these steps:
Sample preparation
First dimension: isoelectric focusing
Second dimension: gel electrophoresis
Staining
Imaging analysis via software

Difference Gel Electrophoresis (DiGE)

Proteome #1 is
labeled with Cy3
(false pink)
Proteome #2 is
labeled with Cy5
(false green)
Mix, run gel, laser
scan for fluorescence