The Hardwiring of development: organization and function of genomic regulatory systems

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

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

3. 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

4. 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

6. 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

7. 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.

8. 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*103 Interference produces distinct phenotype Therefore target site code in CIS region is meaningful at this specificity.

9. 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

10. 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

11. 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

12. 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

14. 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

15. 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

17. 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

18. 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

21. 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