http://cs273a.stanford.edu [Bejerano Aut07/08] 1

MW  11:00-12:15 in Redwood G19Profs: Serafim Batzoglou, Gill BejeranoTA: Cory McLean

http://cs273a.stanford.edu [Bejerano Aut07/08] 2

Lecture 12

Vertebrate Gene Cis-Regulation contd.

http://cs273a.stanford.edu [Bejerano Aut07/08] 3

Vertebrate Gene Regulation

gene (how to)control region(when & where)

DNA

proximal: in 103 letters

distal: in 106 letters

DNA bindingproteins

http://cs273a.stanford.edu [Bejerano Aut07/08] 4

Vertebrate Transcription Regulation

http://cs273a.stanford.edu [Bejerano Aut07/08] 5

unicellular

multicellular

Unicellular vs. Multicellular

http://cs273a.stanford.edu [Bejerano Aut07/08] 6

Pol II Transcription

Key components:• Proteins• DNA sequence• DNA epigenetics

Protein components:• General Transcription factors• Activators• Co-activators

http://cs273a.stanford.edu [Bejerano Aut07/08] 7

Activators & Co-Activators

Protein - DNA

Protein - Protein

http://cs273a.stanford.edu [Bejerano Aut07/08] 8

TFs in the Human Genome

Not a lot…

http://cs273a.stanford.edu [Bejerano Aut07/08] 9

Signal Transduction

http://cs273a.stanford.edu [Bejerano Aut07/08] 10

The Core Promoter

http://cs273a.stanford.edu [Bejerano Aut07/08] 11

CpG islands

http://cs273a.stanford.edu [Bejerano Aut07/08] 12

Cis-Regulatory Components

Low level (“atoms”):• Promoter motifs (TATA box, etc)• Transcription factor binding sites (TFBS)Mid Level:• Promoter• Enhancers• Repressors/Silencers• Insulators/boundary elements• Cis-Regulatory Modules (CRM)• Locus Control Regions (LCR)High Level:• Gene Expression Domains• Gene Regulatory Networks (GRN)

http://cs273a.stanford.edu [Bejerano Aut07/08] 13

Chromatin Remodeling

“off”

“on”

http://cs273a.stanford.edu [Bejerano Aut07/08] 14

Tx Factors Binding Sites

http://cs273a.stanford.edu [Bejerano Aut07/08] 15

Distal Transcription Regulatory Elements

http://cs273a.stanford.edu [Bejerano Aut07/08] 16

Enhancers

http://cs273a.stanford.edu [Bejerano Aut07/08] 17

Basal factors RNAP II

Enhancer with bound protein

promoter

Enhancers: action over very large distances

http://cs273a.stanford.edu [Bejerano Aut07/08] 18

Transient Transgenic Enhancer Assay

Reporter GeneMinimal PromoterConservedElement

Construct is injected into 1 cell embryosTaken out at embryonic day 10.5-14.5Assayed for reporter gene activity

in situ

transgenic

http://cs273a.stanford.edu [Bejerano Aut07/08] 19

Enhancer verification

Matched staining in genital eminence

Matched staining in dorsal apical

ectodermal ridge (part of limb bud)

http://cs273a.stanford.edu [Bejerano Aut07/08] 20

Fly Enhancer Combinatorics

http://cs273a.stanford.edu [Bejerano Aut07/08] 21

Vertebrate Enhancer Combinatorics

http://cs273a.stanford.edu [Bejerano Aut07/08] 22

What are Enhancers?What do enhancers encode?Surely a cluster of TF binding sites.[but TFBS prediction is hard, fraught with false positives]What else? DNA Structure related properties?

So how do we recognize enhancers?Sequence conservation across multiple species[weak but generic]

http://cs273a.stanford.edu [Bejerano Aut07/08] 23

Repressors / Silencers

http://cs273a.stanford.edu [Bejerano Aut07/08] 24

What are Enhancers?What do enhancers encode?Surely a cluster of TF binding sites.[but TFBS prediction is hard, fraught with false positives]What else? DNA Structure related properties?

So how do we recognize enhancers?Sequence conservation across multiple species[weak but generic]

Verifying repressors is trickier [loss vs. gain of function].

How do you predict an enhancer from a repressor? Duh...

repressors

repressorsRepressors

http://cs273a.stanford.edu [Bejerano Aut07/08] 25

Insulators

http://cs273a.stanford.edu [Bejerano Aut07/08] 26

Gene Expression Domains: Independent

http://cs273a.stanford.edu [Bejerano Aut07/08] 27

Gene Expression Domains: Dependent

http://cs273a.stanford.edu [Bejerano Aut07/08] 28

Correlation with Human Disease

[Wang et al, 2000]

http://cs273a.stanford.edu [Bejerano Aut07/08] 29

Other Positional Effects

[de Kok et al, 1996]

http://cs273a.stanford.edu [Bejerano Aut07/08] 30

Chromatin Structure

http://cs273a.stanford.edu [Bejerano Aut07/08] 31

Histone Code

http://cs273a.stanford.edu [Bejerano Aut07/08] 32

Epigenetics

[Goldberg et al, 2007]

http://cs273a.stanford.edu [Bejerano Aut07/08] 33

More Functional Assays

In vitro / in vivoFragment / BACGain / LossBAC cut and paste

http://cs273a.stanford.edu [Bejerano Aut07/08] 34

Protein & Chromatin Assays

Protein binding assays: Electrophoretic mobility shift assays (EMSA) / Gel Shift DNAseI protection SELEX & CASTing Chromatin immuno-precipitation (ChIP), ChIP-chipand various chromatin assays.

http://cs273a.stanford.edu [Bejerano Aut07/08] 35

Gene Regulatory Networks

[Davidson & Erwin, 2006]

http://cs273a.stanford.edu [Bejerano Aut07/08] 36

The Hox Paradox

[Wray, 2003]

http://cs273a.stanford.edu [Bejerano Aut07/08] 37

The Great Vertebrate-Invertebrate Divide

http://cs273a.stanford.edu [Bejerano Aut07/08] 38

Gene Regulatory Network (GRN) Components

Davidson & Erwin (2006): 4 classes of GRN components:• ‘‘kernels’’ evolutionarily inflexible subcircuits that perform

essential upstream functions in building given body parts.• ‘‘plug-ins’’ certain small subcircuits that have been

repeatedly co-opted to diverse developmental purposes(regulatory, inc. signal transduction systems)

• “I/O switches” that allow or disallow developmental subcircuits to function in a given context (e.g., control of size of homologous body parts, many hox genes)

• differentiation gene batteries (execute cell-type specific function, end-players)

http://cs273a.stanford.edu [Bejerano Aut07/08] 39

GRN Kernel properties

1. Network subcircuits that consist of regulatory genes (i.e., TFs).2. They execute the developmental patterning functions required to

specify the embryo spatial domain/s in which body part/s will form. 3. Kernels are dedicated to given developmental functions and are not

used elsewhere in development of the organism (though individual genes of the kernel are likely used in many different contexts).

4. They have a particular form of structure in that the products of multiple regulatory genes of the kernel are required for function of each of the participating cis-regulatory modules of the kernel.

5. Interference with expression of any one kernel gene will destroy kernel function altogether and is likely to produce the catastrophic phenotype of lack of the body part.

The result is extraordinary conservation of kernel architecture.

http://cs273a.stanford.edu [Bejerano Aut07/08] 40

Kernel example

[Davidson & Erwin, 2006]

http://cs273a.stanford.edu [Bejerano Aut07/08] 41

Kernels and Phyla

t

now