[Bejerano Aut07/08] 1 MW 11:00-12:15 in Redwood G19 Profs: Serafim Batzoglou, Gill Bejerano TA: Cory McLean

[Bejerano Aut07/08] 2 Lecture 12 Vertebrate Gene Cis-Regulation contd.

[Bejerano Aut07/08] 3 Vertebrate Gene Regulation gene (how to) control region (when & where) DNA proximal: in 10 3 letters distal: in 10 6 letters DNA binding proteins

[Bejerano Aut07/08] 4 Vertebrate Transcription Regulation

[Bejerano Aut07/08] 5 unicellular multicellular Unicellular vs. Multicellular

[Bejerano Aut07/08] 6 Pol II Transcription Key components: Proteins DNA sequence DNA epigenetics Protein components: General Transcription factors Activators Co-activators

[Bejerano Aut07/08] 7 Activators & Co-Activators Protein - DNA Protein - Protein

[Bejerano Aut07/08] 8 TFs in the Human Genome Not a lot…

[Bejerano Aut07/08] 9 Signal Transduction

[Bejerano Aut07/08] 10 The Core Promoter

[Bejerano Aut07/08] 11 CpG islands

[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)

[Bejerano Aut07/08] 13 Chromatin Remodeling “off” “on”

[Bejerano Aut07/08] 14 Tx Factors Binding Sites

[Bejerano Aut07/08] 15 Distal Transcription Regulatory Elements

[Bejerano Aut07/08] 16 Enhancers

[Bejerano Aut07/08] 17 Basal factors RNAP II Enhancer with bound protein promoter Enhancers: action over very large distances

[Bejerano Aut07/08] 18 Transient Transgenic Enhancer Assay Reporter Gene Minimal Promoter Conserved Element Construct is injected into 1 cell embryos Taken out at embryonic day Assayed for reporter gene activity in situ transgenic

[Bejerano Aut07/08] 19 Enhancer verification Matched staining in genital eminence Matched staining in dorsal apical ectodermal ridge (part of limb bud)

[Bejerano Aut07/08] 20 Fly Enhancer Combinatorics

[Bejerano Aut07/08] 21 Vertebrate Enhancer Combinatorics

[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]

[Bejerano Aut07/08] 23 Repressors / Silencers

[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 Repressors

[Bejerano Aut07/08] 25 Insulators

[Bejerano Aut07/08] 26 Gene Expression Domains: Independent

[Bejerano Aut07/08] 27 Gene Expression Domains: Dependent

[Bejerano Aut07/08] 28 Correlation with Human Disease [Wang et al, 2000]

[Bejerano Aut07/08] 29 Other Positional Effects [de Kok et al, 1996]

[Bejerano Aut07/08] 30 Chromatin Structure

[Bejerano Aut07/08] 31 Histone Code

[Bejerano Aut07/08] 32 Epigenetics [Goldberg et al, 2007]

[Bejerano Aut07/08] 33 More Functional Assays In vitro / in vivo Fragment / BAC Gain / Loss BAC cut and paste

[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-chip and various chromatin assays.

[Bejerano Aut07/08] 35 Gene Regulatory Networks [Davidson & Erwin, 2006]

[Bejerano Aut07/08] 36 The Hox Paradox [Wray, 2003]

[Bejerano Aut07/08] 37 The Great Vertebrate-Invertebrate Divide

[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)

[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.

[Bejerano Aut07/08] 40 Kernel example [Davidson & Erwin, 2006]

[Bejerano Aut07/08] 41 Kernels and Phyla t now