Cell cycle control is fundamental in eukaryotic development. Several modeling efforts have been used to integrate the complex network of interacting molecular components involved in cell cycle dynamics. In this paper, we aimed at recovering the regulatory logic upstream of previously known components of cell cycle control, with the aim of understanding the mechanisms underlying the emergence of the cyclic behavior of such components. We focus on Arabidopsis thaliana, but given that many components of cell cycle regulation are conserved among eukaryotes, when experimental data for this system was not available, we considered experimental results from yeast and animal systems. We are proposing a Boolean gene regulatory network (GRN) that converges into only one robust limit cycle attractor that closely resembles the cyclic behavior of the key cell-cycle molecular components and other regulators considered here. We validate the model by comparing our in silico configurations with data from loss- and gain-of-function mutants, where the endocyclic behavior also was recovered. Additionally, we approximate a continuous model and recovered the temporal periodic expression profiles of the cell-cycle molecular components involved, thus suggesting that the single limit cycle attractor recovered with the Boolean model is not an artifact of its discrete and synchronous nature, but rather an emergent consequence of the inherent characteristics of the regulatory logic proposed here. This dynamical model, hence provides a novel theoretical framework to address cell cycle regulation in plants, and it can also be used to propose novel predictions regarding cell cycle regulation in other eukaryotes. Bird Shannyn birdgirl@arcticmail.com University of Nebraska-Lincoln 2016-09-12T08:51:56Z 2017-10-13T13:09:34Z

Anaphase-promoting complex/Cyclosome

 2017-10-12T16:10:40Z

Protein name: Cyclin-B1-1

Gene name: CYCB1-1

UniProt ID: P30183

Gene ID: 829904

 2017-10-12T16:11:01Z

Protein name: Cyclin-D3-1

Gene name: CYCD3-1

UniProt ID: P42753

Gene ID: 829564

 2017-10-12T21:30:02Z

Protein name: MYB77

Gene name: AtMYB77

UniProt ID: O49745

Gene ID: 824168

 2017-10-12T15:59:44Z

Protein name: E2F transcription factor-like E2FE

Gene name: E2FE

UniProt ID: Q8LSZ4

Gene ID: 823971

 2017-10-12T15:59:44Z

Skp1/Cullin/F-box

 2017-03-24T12:52:43Z

Protein name: Cyclin-dependent kinase inhibitor 1

Gene name: KRP1

UniProt ID: Q67Y93

 2017-10-12T21:30:02Z

Protein name: Transcription factor E2FB

Gene name: E2FB

UniProt ID: Q9FV71

Gene ID: 832283

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Protein name: Cyclin-A2-3

Gene name: CYCA2-3

UniProt ID: Q38819

Gene ID: 838127

 2017-10-12T16:11:01Z

Protein name: Transcription factor E2FA

Gene ID: 818174

Gene name: E2FA

UniProt ID: Q9FNY0

 2017-07-18T14:27:47Z

Protein name: Myb-related protein 3R-1/MYB transcription factor

Gene name: MYB3R-1/MYB3R4

UniProt ID: Q9S7G7/Q94FL9

Gene ID: 829409/831023

 2017-10-12T16:11:01Z

Protein name: Retinoblastoma-related protein 1

Gene name: RBR1

Uniprot ID: Q9LKZ3

Gene ID: 820408

 2017-10-12T16:11:01Z

Retinoblastoma-related protein 1

Gene name: RBR1

UniProt ID: Q9LKZ3

Gene ID: 820408

 2017-10-12T15:59:44Z

Protein name: Transcription factor E2FC

Gene name: E2FC

UniProt ID: Q9FV70

Gene ID: 841202

 2017-10-12T15:59:44Z

E2Fa activates APC/C.

E2Fe inhibits APC/C.

MYB3R1/4 activates APC/C.

MYB77 activates APC/C.

RBR inhibits APC/C.

MYB transcription factors are important for the G2/M transition. APC/C plays a role in mitosis exit. In this model it is proposed that MYB77 is a transcription factor that links, either directly of indirectly, the start of M phase to the end of M phase through upregulation of APC/C.

E2Fa activates CCS5A2, a component of APC/C.

RBR is important in progressing the cell through G1. RBR activity implies that mitosis has ended and APC/C is no longer needed by the cell. Thus in this model it is poposed that RBR activity inhibits, either directly or indirectly, APC/C.

SKP2A, RBR, CDKB1;1, CYCA2;3, CCS52A2, KRP1, E2Fc, MYB3R1/4 and CYCB1;1 were found to contain consensus sites for MYB3R4.

E2F2 downregulates CCS5A2, a component of APC/C.

 S_10 1 S_5 1 S_13 1 S_11 1 S_5 1 S_4 1 S_5 1

MYB3R1/4 activates CYCB1;1.

KRP1 in conjunction with CYCD3;1 inhibits CYCB1;1.

APC/C inhibits CYCB1;1.

MYB77 activates CYCB1;1.

RBR inhibits CYCB1;1.

E2Fb activates CYCB1;1.

E2Fc inhibits CYCB1;1.

The APC/C complex ubiquitinates CYCB1;1, leading to its subsequent degradation.

E2Fc expression reduces CDKB1;1 levels.

MYB77 recognizes the sequence CNGTTR as a consensus site. This information was used to find possible targets in the cell cycle.

Expression of E2Fb induces CYCB1;1 expression.

CyCD3;1, an indirect activator of CYCB1;1, is absent or inactive when KRP1 is present.

RBR represses E2Fb activity. Phosphorylation of RBR in G1 leads to an accumulation of E2Fb.

MYB3R1/4 recognizes the sequence AACGG found on CYCB1;1.

KRP1 binds to CDKA;1 to inhibit the activity of the CDKA–CYCB1;1 complex.

 S_4 1 S_1 1 S_8 1 S_13 1 S_3 1 S_7 1 S_14 1 S_1 1 S_11 1 S_1 1

SCF inhibits CYCD3;1.

SCF is involved in the ubiquitination and subsequent degradation of CYCD3;1.

 S_6 1

E2Fb activates MYB77 when CYCD3;1 is active and KRP1 is inactive or when RBR is inactive.

MYB77 is a predicted mediator between E2F and MYB. E2F has a binding site that is recognized by MYB77.

CYCD3;1 is required to progress the cell through G1, which activates E2Fb.

RBR represses E2Fb activity. Phosphorylation of RBR in G1 leads to an accumulation of E2Fb.

CyCD3;1, an indirect activator of MYB77, is absent or inactive when KRP1 is present.

 S_8 1 S_3 1 S_7 1 S_13 1

E2Fb activates E2Fe.

CYCD3;1 activates E2Fe when KRP1 is inactive.

MYB77 activates E2Fe.

E2Fc inhibits E2Fc.

E2Fc inhibits gene that promote cell division. In this model the absence of E2Fc is sufficient to activate E2Fe.

MYB77 recognizes the sequence CNGTTR as a consensus site. This information was used to find possible targets in the cell cycle.

E2Fb activates E2Fe transcription.

CYCD3;1 is required to progress the cell through G1, which activates E2Fb.

RBR inhibits E2Fb targets.

CyCD3;1, an indirect activator of E2Fe, is absent or inactive when KRP1 is present.

 S_3 1 S_14 1 S_4 1 S_8 1 S_13 1 S_7 1 S_3 1 S_8 1 S_13 1 S_14 1 S_4 1 S_7 1 S_3 1 S_8 1 S_13 1 S_7 1 S_14 1 S_4 1 S_8 1 S_14 1 S_7 1 S_4 1 S_3 1 S_13 1 S_8 1 S_7 1 S_14 1 S_4 1 S_3 1 S_13 1 S_8 1 S_13 1 S_14 1 S_4 1 S_3 1 S_7 1 S_13 1 S_14 1 S_4 1 S_8 1 S_3 1 S_7 1 S_8 1 S_14 1 S_4 1 S_3 1 S_13 1 S_7 1 S_4 1 S_3 1 S_13 1 S_7 1 S_14 1 S_4 1 S_8 1 S_8 1 S_13 1 S_7 1 S_3 1 S_8 1 S_13 1 S_7 1 S_14 1 S_4 1 S_3 1 S_3 1 S_7 1 S_14 1 S_4 1 S_8 1 S_13 1 S_3 1 S_8 1 S_7 1 S_14 1 S_4 1 S_13 1 S_13 1 S_7 1 S_14 1 S_4 1 S_8 1 S_3 1 S_3 1 S_13 1 S_14 1 S_4 1 S_8 1 S_7 1 S_7 1 S_14 1 S_4 1 S_8 1 S_3 1 S_13 1 S_14 1 S_4 1 S_8 1 S_3 1 S_13 1 S_7 1

E2Fb activates SCF when CYCD3;1 is active and RBR is inactive or when RBR is inactive.

MYB3R1/4 activates SCF.

APC/C inhibits SCF.

The APC/C subunit Cdh1 aids in the degradation of Skp2, a subunit of SCF.

The F-box protein FBL17 is regulated by E2F factors.

CYCD3;1 is required to progress the cell through G1, which activates E2Fb.

RBR represses E2Fb activity. Phosphorylation of RBR in G1 leads to an accumulation of E2Fb.

SKP2A, RBR, CDKB1;1, CYCA2;3, CCS52A2, KRP1, E2Fc, MYB3R1/4 and CYCB1;1 were found to contain consensus sites for MYB3R4.

CyCD3;1, an indirect activator of SCF, is absent or inactive when KRP1 is present.

 S_11 1 S_1 1 S_8 1 S_13 1 S_3 1 S_7 1 S_1 1

CDKB1;1 in conjunction with CYCA2;3 and SCF inhibits KRP1.

MYB77 activates KRP1.

MYB3R1/4 activates KRP1.

CDKB1;1 in complex with CYCA2;3, promotes proteolysis of KRP1.

CDKB1;1 in complex with CYCA2;3, promotes proteolysis of KRP1.

MYB77 recognizes the sequence CNGTTR as a consensus site. This information was used to find possible targets in the cell cycle.

SKP2A, RBR, CDKB1;1, CYCA2;3, CCS52A2, KRP1, E2Fc, MYB3R1/4 and CYCB1;1 were found to contain consensus sites for MYB3R4. KRP1 may be induced by MYB3R1/4.

SCF ubiquitinates KRP1, leading to its degradation.

 S_4 1 S_12 1 S_9 1 S_6 1 S_11 1 S_12 1 S_9 1 S_6 1

RBR inhibits E2Fb.

E2Fa activates E2Fb.

E2Fa activates E2Fb transcription.

RBR inhibits E2Fb.

 S_10 1 S_13 1

MYB77 activates CYCA2;3.

MYB3R1/4 activates CYCA2;3.

APC/C inhibits CYCA2;3.

CYCA2;3 is stabilized by a loss-of-function mutation in the APC/C complex.

MYB77 recognizes the sequence CNGTTR as a consensus site. This information was used to find possible targets in the cell cycle.

SKP2A, RBR, CDKB1;1, CYCA2;3, CCS52A2, KRP1, E2Fc, MYB3R1/4 and CYCB1;1 were found to contain consensus sites for MYB3R4.

 S_4 1 S_1 1 S_11 1 S_1 1

E2Fc inhibits E2Fa.

E2Fa activates E2Fa.

CDK1;1 in conjunction with CYCA2;3 inhibits E2Fa.

CDKB1;1 and CYCA2;3 are listed as positive regulators when the other three nodes are inactive in order to match the Boolean logic E2Fa = (E2Fa ∨ ¬E2Fc) ∧ ¬(CDKB1; 1 ∧ CYCA2; 3). These interactions are added in order to match the behavior in the original equation.

E2Fa mRNA increases when E2Fc is silenced.

EF2 has a CDK-phosphorylation sites. Additionally, high CDK activity inversely correlates with E2Fa DNA binding activity.

EF2 has a CDK-phosphorylation sites. Additionally, high CDK activity inversely correlates with E2Fa DNA binding activity.

A positive feedback loop (modeled here as a self-feedback loop) amplifies E2Fa activity.

 S_9 1 S_14 1 S_12 1 S_10 1 S_10 1 S_12 1 S_9 1 S_12 1 S_14 1 S_9 1 S_10 1 S_14 1 S_9 1 S_12 1 S_10 1

MYB3R1/4 in conjunction with CYCB1;1 actuvates MYB3R1/4.

MYB77 activates MYB3R1/4.

KRP1 inhibits MYB3R1/4.

MYB77 recognizes the sequence CNGTTR as a consensus site. This information was used to find possible targets in the cell cycle.

SKP2A, RBR, CDKB1;1, CYCA2;3, CCS52A2, KRP1, E2Fc, MYB3R1/4 and CYCB1;1 were found to contain consensus sites for MYB3R4.

Coexpression of CYCB1;1 and MY3R4 enhances activity of MY3R4 targer promoters by 4-fold.

KRP1 indirectly inhibits MYB3R1/4.

Coexpression of CYCB1;1 and MY3R4 enhances activity of MY3R4 targer promoters by 4-fold.

 S_4 1 S_11 1 S_2 1 S_7 1

MYB77 activates CDKB1;1.

E2Fb activates CDKB1;1 when KRP1 is inactive and CYCD3;1 is active or when RBR is inactive.

MYB3R1/4 activates CDKB1;1.

E2Fc inhibits CDKB1;1.

E2Fc destabilizes CDKB1;1.

MYB77 recognizes the sequence CNGTTR as a consensus site. This information was used to find possible targets in the cell cycle.

Expression of E2Fb promotes CDKB1;1 expression.

CYCD3;1 is required to progress the cell through G1, which activates E2Fb. CyCD3;1 is absent or inactive when KRP1 is present.

RBR represses E2Fb activity. Phosphorylation of RBR in G1 leads to an accumulation of E2Fb.

SKP2A, RBR, CDKB1;1, CYCA2;3, CCS52A2, KRP1, E2Fc, MYB3R1/4 and CYCB1;1 were found to contain consensus sites for MYB3R4.

CyCD3;1, an indirect activator of CDKB1;1, is absent or inactive when KRP1 is present.

 S_4 1 S_8 1 S_13 1 S_3 1 S_7 1 S_14 1 S_11 1

E2Fa (when KRP1 is active and CYCD3;1 is inactive) activates RBR.

MYB3R1/4 (when KRP1 is active and CYCD3;1 is inactive) activates RBR.

RBR (when KRP1 is active and CYCD3;1 is inactive) inhibits RBR.

E2Fa upregulates RBR transcription.

Complexes formed by CDKA;1 and D-type cyclins inactivate RBR via phosphorylation.

A negative feedback loop (modeled as a self feedback loop) regulates RBR activity.

SKP2A, RBR, CDKB1;1, CYCA2;3, CCS52A2, KRP1, E2Fc, MYB3R1/4 and CYCB1;1 were found to contain consensus sites for MYB3R4.

CyCD3;1 phosphorylates and inhibits RBR. CyCD3;1 is absent or inactive when KRP1 is present.

 S_11 1 S_7 1 S_3 1 S_10 1 S_7 1 S_3 1 S_13 1 S_3 1 S_7 1

SCF (when KRP1 is inactve and CYCD3;1 is active) inhibits E2Fc.

MYB3R1/4 activates E2Fc.

E2Fa binds to and activates E2Fc.

RBR inhibits E2Fc.

E2Fa binds to and activates E2Fc.

CDKA;1 bound to D-type cyclin phosphorylates E2Fc so that it can be recognized by the SCF complex.

RBR inhibits E2Fc.

SKP2A, RBR, CDKB1;1, CYCA2;3, CCS52A2, KRP1, E2Fc, MYB3R1/4 and CYCB1;1 were found to contain consensus sites for MYB3R4.

SKP2A is a subunit of SCF. SKP2A overexpression reduces E2Fc levels.

CyCD3;1, an indirect activator of E2Fc, is absent or inactive when KRP1 is present.

 S_11 1 S_6 1 S_3 1 S_7 1 S_10 1 S_6 1 S_3 1 S_7 1 S_13 1