Transcript Organization of Cis-regulatory Systems

The Hardwiring of development: organization
and function of genomic regulatory systems
Maria I. Arnone and Eric H. Davidson
Outline:
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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*103
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-typespecific 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