A functional and evolutionary perspective on transcription factor binding in Arabidopsis thaliana Klaas Vandepoele Comparative & Integrative Genomics group Department of Plant Biotechnology and Bioinformatics, Ghent University Department of Plant Systems Biology, VIB - Belgium

1. A functional and evolutionary perspective on
transcription factor binding in Arabidopsis thaliana
Potsdam, October 2014
Comparative & Integrative Genomics group
Department of Plant Biotechnology and Bioinformatics, Ghent University
Department of Plant Systems Biology, VIB - Belgium
plaza_genomics

2. OVERVIEW
1. Transcriptional gene regulation in plants
2. Inference of transcriptonal networks using an ensemble
framework for phylogenetic footprinting in plants
3. An integrated gene regulatory network using experimental ChIP
data of 27 transcription factors in Arabidopsis
4. Conclusions
Jan Van de Velde
Ken Heyndrickx

3. 1. TRANSCRIPTIONAL GENE REGULATION
Mejia-Guerra et al., 2012
Arabidopsis
-1,700-2,500 Transcription Factors
- 180-791 miRNA
- 2,708 expressed lncRNA
49MB non-coding DNA
11,000 regulatory
interactions (AtRegNet)

4. EXPERIMENTAL CHARACTERIZATION OF REGULATORY
INTERACTIONS
Mejia-Guerra et al., 2012
EMSA
Y1H
SELEX
PBM
ChIP-Seq

5. COMPUTATIONAL ANALYSIS OF CIS-REGULATORY
ELEMENTS
 Mapping of known TF binding sites on promoter sequences
 False positives
 Low quality motifs (PWMs) + many motifs lack information about
binding factor
 Motif redundancy & multi-gene transcription factor families
Database # CRE Species
PLACE 469 Vascular plants
AGRIS 99 Arabidopsis thaliana
AtProbe 172 Arabidopsis thaliana
PlantCARE 435 Monocots and dicots

6. 2. PHYLOGENETIC FOOTPRINTING: DETECTION OF
CONSERVED NON-CODING SEQUENCES (CNS)
 Comparative analysis of noncoding DNA sequences to identify
candidate regulatory elements (in orthologous genes)
 Regulatory elements are conserved during evolution due to
functional constraint (vs. neutral carry-over)
 The power of phylogenetic footprinting is enhanced significantly
when data from a number of related species, which diverged
sufficiently, is available

7. DEVELOPING AN ENSEMBLE FRAMEWORK FOR
PHYLOGENETIC FOOTPRINTING IN PLANTS
 Application of motif mapping and
different pairwise alignment tools
 Aggregate alignments in multi-
species footprint using 11
comparator dicot genomes
 Evaluate statistical signifcance incl.
FDR analysis
AtProbe
Feature map
@ RSAT
144 regulatory elements (63 genes)
774 DNA motifs

8. FROM PAIRWISE ALIGNMENTS TO MULTI-SPECIES FOOTPRINTS
 Generate all pairwise alignments
between Arabidopsis query gene
and its orthologs
 Map all pairwise alignments back
to reference promoter
 Count per position the #species
that support a footprint
 Significance estimation
Van Bel, M., Proost, S., Wischnitzki, E., Movahedi, S., Scheerlinck, C., Van de Peer, Y., Vandepoele, K. (2012) Dissecting plant
genomes with the PLAZA comparative genomics platform. Plant Physiology 158:590-600

9. EVALUATION ATPROBE EXPERIMENTAL CIS-
REGULATORY ELEMENTS
Significance
Experimental
motifs
Scmm ACGTGGC = 0.54
P value < 0.001
G-box
Scmm ATAGATAA = 0.09
P value 0.48
GA motif
Scmm GATAAGATT = 0.36
P value < 0.001
I-box
RBCS1A
Scmm TATATATA = 0.7
P value < 0.001
GAPA
ACA motif
C-motif

10. PROPERTIES CNS
 69,361 CNSs associated with 17,895 genes
 Protein-coding genes (99%), miRNA genes (1%)
 Median length: 11nt (min-max: 5-514nt)
 CNS cover 1,070kb of the non-coding Arabidopsis genome

11. DETECTION OF EXPERIMENTAL REGULATORY
ELEMENTS
• Black boxes: percentage of
recovered elements
• White boxes: percentage
of uniquely recovered
elements in this study

12. RECOVERY OF IN VIVO FUNCTIONAL TARGETS
USING CNS INFORMATION
• White boxes: fold enrichment for CNSs
• Black boxes: fold enrichment naïve motif mapping
High-quality dataset 15 TFs
ChIP-Seq binding + TF binding site + differentially expressed TF perturbation (n=2708)

13. GENOME-WIDE REGULATORY ANNOTATION
Collapsed TF-target module network
 40,758 TF-target interactions (157 TFs)
 9/13 TFs significant overlap with experimentally
confirmed targets (AtRegNet/Hussey et al., 2013)
 Various functional genomics metrics confirm quality
predicted GRN
Van de Velde, J.*, Heyndrickx, K.S.*, and Vandepoele, K. (2014). Inference of Transcriptional Networks in Arabidopsis through
Conserved Noncoding Sequence Analysis. Plant Cell.

14. A CONDITION-SPECIFIC SECONDARY CELL WALL
GENE REGULATORY NETWORK.

15. 2. AN INTEGRATED GENE REGULATORY NETWORK USING CHIP
DATA OF 27 TRANSCRIPTION FACTORS
 How is TF binding organised across different target genes?
 Have Highly Occupied Target (HOT) genes in plants the same
distinct regulatory features as in organisms?
 To what extent is binding linked to differential expression TF
binding site presence?

16. * Heyndrickx KS, Vandepoele K (2012) Systematic Identification of Functional Plant Modules through the Integration of Complementary Data
Sources. Plant physiology 159: 884-901

17. TF PROTEIN-PROTEIN INTERACTION NETWORK
De Bodt S, Hollunder J, Nelissen H, Meulemeester N, Inze D (2012) CORNET 2.0: integrating plant coexpression, protein-protein
interactions, regulatory interactions, gene associations and functional annotations. The New phytologist 195: 707-720
Flowering
Light Response

18. GENOMIC REGIONS BOUND BY TFS

19. GENOMIC REGIONS BOUND BY TFS

20. GENE LOCATION ANALYSIS TF BINDING EVENTS
 89% of upstream sites lie < 2kb
 23,891 / 26,717
 91% of downstream sites lie < 2kb
 11,687 / 12,828

21. COORDINATED REGULATION BY DIFFERENT TFS

22. BINDING SITE ORGANISATION
A
B
DH I sites: Zhang W, Zhang T, Wu Y, Jiang J (2012) Genome-Wide Identification of Regulatory DNA Elements and Protein-Binding
Footprints Using Signatures of Open Chromatin in Arabidopsis. The Plant cell
A
B
C
D
R

23. COOPERATIVE TF BINDING: HUB & HOT
#TargetGenes
# Bound TFs
Gene Complexity
A-D

24.  Hub genes
 1,170 genes bound by ≥ 8 TFs
 Significantly Enriched for TFs and miRNAs
 Highly Occupied Target (HOT) regions
 1,179 regions bound by ≥ 7 TFs
COOPERATIVE TF BINDING: HUB & HOT
A-D
Enrichment for regulatory genes
(TFs, kinases), response to
stimuli & developmental genes

25. CHROMATIN STATES AND NUCLEOTIDE DIVERSITY OF TF-
BOUND REGIONS
Sequeira-Mendes et al., … Gutierrez, C. (2014). The Functional Topography of the Arabidopsis Genome Is Organized in a Reduced
Number of Linear Motifs of Chromatin States. Plant Cell.
Population sequence diversity based on
369 Arabidopsis strains (Weigel lab)

26. HOT-ASSOCIATED GENES ARE FACTOR-RESPONSIVE

27. HOT-ASSOCIATED GENES ARE FACTOR-RESPONSIVE

28. EXPRESSION LEVELS ARE CORRELATED WITH
THE TOTAL NUMBER OF BOUND TFS
Low: < 3 TFs; Intermediate: >= 3 TFs and < 8TFs; hub: >= 8 TFs
(n=406 flowering-related genes)

29. A SINGLE MOTIF DOES NOT EXPLAIN BINDING

30. A SINGLE MOTIF DOES NOT EXPLAIN BINDING (II)

31. A SINGLE MOTIF DOES NOT EXPLAIN BINDING (II)

32. SEC. MOTIFS ARE ENRICHED FOR DE AS WELL
PIF5: ChIP-Seq

33. SEC. MOTIFS ARE ENRICHED FOR DE AS WELL

34. NEW HYPOTHESES ON CO-BINDING AND
TETHERING
AP1
PIF3
PRR7
FHY3AP1 SEP3
Co-binding Tethering
PRR5 PRR7

35. CONCLUSIONS & PERSPECTIVES
 Integration of CNS with complementary experimental data sources
offers new possibilities for regulatory gene annotation in plants
 High specificity to predict TF-target interactions
 Complementary to exerperimental TF-target detection methods
 Study GRN conservation and rewiring across species
 Integrated 27 TF ChIP-Seq gene regulatory network reveals
 Complexly regulated are enriched for regulatory genes
 HOT-associated regions represent functional binding events
 Open chromatin
 Sequence constraint
 TF binding sites
 Enrichment for regulated target genes
 Co-binding and tethering patterns could explain the apparent
discrepancy between binding and regulation in ChIP-chip/Seq
studies

36. FURTHER READING
Van de Velde, J.*, Heyndrickx, K.S.*, and Vandepoele, K. (2014). Inference of
Transcriptional Networks in Arabidopsis through Conserved Noncoding Sequence
Analysis. The Plant Cell 26(7):2729-2745
Proost, S., Van Bel, M. … and Vandepoele, K. (2015). PLAZA 3.0: an access point
for plant comparative genomics. Nucleic Acids Research (accepted)
Heyndrickx, K.S.*, Van de Velde, J.*, and Vandepoele, K. (2014). A functional
and evolutionary perspective on transcription factor binding in Arabidopsis
thaliana. The Plant Cell (accepted)