The Hardwiring of development: organization and function of genomic regulatory systems Maria I. Arnone and Eric H. Davidson

Outline: Introduction Properties of CIS-Regulatory Systems Gene Regulatory Networks Conclusion and Summary

Introduction Regulatory program is hardwired into genomic DNA sequence CIS-regulatory target sites are recognized sequences-specifically by T.F Control Rate, activation, repression, time and morphological space Reveals internal working and interconnections i.e., structure of gene regulatory network

CIS-regulatory systems are complex Objective is to understand the flow of regulatory information from the genome to understand development Inputs and outputs studied…., but… To find out how genomic regulatory systems really work, examination of the CIS sequence themselves is necessary

Properties of CIS-Regulatory Systems Regulatory modules: Separate CIS elements, multiple T.F target sites Can transmit output to BTA Communication achieved by T.F anchored or proteins that bind T.F Long distance achieved by DNA looping Works with promoters i.e. platform on which BTA assembles Novel expression constructs can be created

Intramodular Complexity Complexity is number of diverse interactions, i.e., T.F bound per module Most cases complexity is underestimated Avg. # of interactions is 6.2 Factors are of diverse chemical nature, no modules serviced by only homeodomain proteins or Zn finger proteins.

Significance of DNA-protein interactions detected in vitro Does binding imply significance? Three reasons –Equilibrium constants –When affinity is low, cooperative interactions stabilize it –Length of sequence protected by bound T.F specify these sites uniquely Various experiments done to show that sequence preference of T.F for its target site is at least 5-10*10 3 Interference produces distinct phenotype Therefore target site code in CIS region is meaningful at this specificity.

Positive and Negative functions of factors binding within cis-regulatory modules Modular elements target of T.F with diverse functions Focus on activation and repression Maternal factors in embryo Mutation in repressor modules => ectopic expression Mutation in activator target sites => lower level of normal expression Activators and repressors act in conjunction to regulate genes

Programming communication within complex cis-regulatory systems Many CIS regulatory elements interact at both near and far distances, ex: by forming loops Dwell time for the complexes is 15-80mins Proximal CIS-regulatory modules process outputs of distally located modules Interactions increase diversity of control functions of CIS system Looping occurs due to affinity of proteins Intermodular communication is therefore hardwired

Cis-regulatory organization as an index of developmental role Goal is to use cis-regulatory target sites to predict role in regulation and position in network. Modules that interpret embryonic spatial specification bind activators and repressors Ones that use only positive regulators likely to act downstream T.F confined to spatial domains and serve as activators in downstream genes in particular regions CIS regulators of these genes may operate by activation as well as repression

Gene Regulatory Networks Linkage between different cis-regulatory systems together with genes that they govern Batteries of genes that encode cell-type- specific differentiation proteins Linkage between genes that encode T.F and differentiation proteins

Gene batteries Sets of genes expressed in different stages of development Genes that are coordinately expressed because their cis-regulatory sequences share homologous sites for TF But, TF for which genes of a battery share sites are not cell-type specific Order and spacing of target sites not alike in any two genes of same cell type Reflects lack of functions constraint i.e. spacing and order Battery relations can only be elucidated by direct analysis Analysis of batteries will help to go from protein of known function to interior of gene regulatory network

General Considerations Peripheral and internal network elements: –Linkage to CIS only upstream or both upstream and downstream i.e. TF Network and Casual relationships: -Refer to figure Multilevel connections: -Downstream connections from internal genes to peripheral genes -Autoregulatory connections -Therefore network cannot be considered hierarchical

View from the genome and View from the nucleus Figures show view from genome View from nucleus describes developmental state CIS region may be fully loaded, partially loaded or empty depending on state of cell Per-genome networks have no temporal dependence Per-nuclear networks depend on time

Complexity of developmental gene regulation # of linkages upstream from a cis-regulatory system Integration of spatial information to regulate TF during growth and cellular expansion Complex network linkages even in early developmental stages Initially transcribed peripheral genes from maternal effect may have shallow regulatory network and therefore may be within reach for regulatory analysis

Conclusion Importance of cis-regulatory analysis Path to understand the organization of the genomic program for development. Analyzing networks will lead to solving developmental and evolutionary questions Primary genomic sequence data will be most important