# Prediction of Time Series Gene Expression and Structural Analysis of Gene Regulatory Networks Using Recurrent Neural Networks

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## Abstract

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## 1. Introduction

## 2. Materials and Methods

#### 2.1. Dual Attention Mechanism Structure and Learning Procedures

#### Choice of the Time Window for Network Training

#### 2.2. Deep Neural Network Parameter Analysis

#### 2.3. Response of the Prediction to Noise Addition

#### 2.4. Clustering and Principal Component Analysis

## 3. Results

#### 3.1. Gene Regulatory Network Dynamic

- ${J}_{ij}$ is the interaction matrix among the genes i and j, which is 1 if gene j expresses a protein, at concentration ${x}_{j}$, that is a TF for gene i, −1 if it is a repressor and 0 if the two genes do not interact.
- ${k}_{ij}$ and ${\tilde{k}}_{ij}$ are the expression and repression rates for the gene i from the TF (repressor) j.
- ${h}_{ij}$ is the cooperativity index of the $ij$ interaction.
- ${K}_{ij}$ is the dissociation constant of the reaction $ij$.
- ${\tau}_{ij}$ is the delay between the expression of the protein ${x}_{j}$ and the time when it is ready to activate (repress) the gene i. It allows one to account for the translation of mRNAs into proteins and it has been previously included in Gillespie simulations [50,51,52]. However, in the present study, we set it to zero for the sake of simplicity.

#### 3.2. Recurrent Neural Network for Stochastic Time Traces Generated by Different Gene Regulatory Network Architectures

#### 3.3. Studying the Input Attention Mechanism and the Response of the Prediction to Noise

#### 3.4. Network Properties of the Input Attention Distinguishing Gene Regulatory Network Architectures

#### 3.5. Differential Response to Noise in the Prediction of Time Series Gene Expression Data

#### 3.6. Comparison between Clustering of Gene Regulatory Networks

## 4. Discussion

## Supplementary Materials

## Author Contributions

## Funding

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

## Appendix A

- Define the network, the matrix of interaction, the nature of the interactions, the rates of the reactions, the number of species (nodes), the final time ${T}_{max}$ of the simulation and the propensity functions ${a}_{j}$;
- Select the time of the first reaction from the exponential probability distribution of the times, $P\left(\tau \right)={e}^{-{a}_{tot}\tau}$;
- Select the reaction from the relative propensity, $P\left(j\right)=\frac{{a}_{j}}{{a}_{tot}}$;
- Upgrade the species according to the rules of the reaction j;
- Compute the new propensities ${a}_{j}$ and ${a}_{tot}={\sum}_{j}{a}_{j}$ from the updated set of the network;
- Update the time $t+=\tau $;
- Repeat from step 1 until $t<{T}_{max}$.

## Appendix B

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**Figure 1.**(

**A**): Scheme of the dual attention recurrent neural network (DA-RNN) used in this study. From left to right: The network was trained on gene expression time traces of length T. The first attention layer (input attention) encoded which input time traces were more important for target prediction. The second attention layer (temporal attention) encoded the most important time points of the input time traces for the prediction. The LSTM chain on the right was the decoder that provided the final output. (

**B**): Architecture of the parallel DA-RNN. Each box represents a DA-RNN devoted to the prediction of gene i; after the prediction of the next time step, the data were collected to fuel the inputs for the parallel nets and to predict the next time point. (

**C**): Distribution of the autocorrelation time for the genes of a fully connected and an oscillating network (see Section 3.2 for details on different GRN architectures). (

**D**): Training time for all the genes as a function of the time window T used for training, for the same two networks. (

**E**): RMSE as a function of the time window T used for training, for the same two networks. (

**F**): Kullback–Leibler divergence (circles) and Jensen–Shannon distance (triangles) between the probability distribution of the real data and that of the predicted ones, plotted as a function of the training window, for the same two networks. The values shown for each value of the time window are averages over all the genes of the GRN.

**Figure 2.**Gene regulatory network dynamic with $N=3$ genes. The dynamic was generated using a random interaction matrix ${J}_{ij}$. The top panel shows the continuous time trace of proteins concentration for a random GRN with ${n}_{r}={n}_{a}=0.2$. The bottom panel shows the oscillatory dynamic of 3 genes relative to the core clock of an oscillatory network. In both panels, dots show the prediction of the neural network implemented using the first $T=10$ time points and then letting the system evolve on its own. The stochastic data were generated using the Gillespie algorithm.

**Figure 3.**Time dynamics and full probability function (integrated over all the time of the simulation) of having a protein at a certain concentration. We show selected example genes from an oscillatory GRN (panels

**B**,

**D**) and a GRN controlled by a master regulator gene (panels

**A**,

**C**), both with ${n}_{r}={n}_{a}=0.5$. The plots show the performance of the prediction comparing the time traces between the stochastic simulation and the DNN propagation. Moreover, the resulting probability distribution of the given protein concentration for all times is reported. We highlight that the protein concentration dynamic was reconstructed with high accuracy and even the noise amplitude and the mean were quite well reported by the neural net. Moreover, for the genes belonging to the oscillating GRN (panels

**B**,

**D**), it was possible to reconstruct the amplitude and the mean of the oscillations. We stress that the dynamic shown in panel

**B**could not be immediately addressed to a gene driven by an oscillatory clock, but the bi-modal distribution shows that this was the case. Interestingly, this reveals that, given a model trained on real data, we can look at its internal parameters that allow us to determine if the genes in the GRN belong or not to a certain network topology such as an oscillatory-driven network, such as, for instance, the cell cycle or circadian clock.

**Figure 4.**Comparison of the matrices resulting from the input attention and response-to-noise analyses. Scatter plots showing the relationship between the matrix elements of those obtained computing the network properties of the input attention (clustering coefficient, hub score and betweenness) and the matrix of normalized mean MSE obtained from the analysis of the response of the prediction to noise. Note that the data are represented in log–log scale using the transformation $sign\left(x\right)ln(1+|x\left|\right)$, since they included both positive and negative values. The Pearson correlation coefficient $\rho $ between each pair of properties, computed on the transformed data, is reported on top of each panel.

**Figure 5.**Clustering based on the network properties of the input attention matrices. We show the dendrograms obtained from a hierarchical clustering (

**left**), a PCA plot showing the 12 gene regulatory networks used (

**centre**) and the obtained partition in groups (

**right**) for the clustering coefficient (

**A**), betweenness (

**B**) and hub score (

**C**).

**Figure 6.**Impact of noise addition on time series gene expression prediction for different GRN architectures. (

**A**): Matrices of the mean square error (MSE) on the prediction by the DA-RNN, for an oscillating GRN with 10 repressors and activators per gene on average (

**left**) and a GRN controlled by a master regulator (

**right**). Rows (genes) are ranked according to the mean of the MSE (last column). (

**B**): Dendrogram obtained from the hierarchical clustering of the matrix summarizing the response of the prediction to noise for each gene regulatory network. (

**C**,

**D**): PCA showing the 12 GRNs used and their partition in clusters.

**Figure 7.**Comparison between the clusterings. (

**A**): Comparison between the dendrograms of the different hierarchical clustering shown in Figure 5 and Figure 6 using the information-based generalized Robinson–Foulds distance (or tree distance). (

**B**): Comparison between the partitions obtained using the variation of information.

**Table 1.**Prediction accuracy of the DA-RNN for different gene regulatory network architectures. The network types are those described in Section 3.2, where ${n}_{r}$ and ${n}_{a}$ indicate the mean number of repressors and activators per gene, respectively. We report the mean and standard deviation of the root-mean-square error (RMSE) computed over all the genes in each network.

Network | Mean (RMSE) | Std (RMSE) |
---|---|---|

FullyConnected | 0.12 | 0.04 |

FullyRepressed | 0.67 | 0.08 |

MasterRegulator | 0.83 | 0.04 |

SparseConnection | 0.14 | 0.05 |

mediumConnection | 0.35 | 0.23 |

Oscillating_nr1_na1 | 0.22 | 0.11 |

Oscillating_nr5_na5 | 0.23 | 0.13 |

Oscillating_nr10_na10 | 0.30 | 0.17 |

Oscillating_nr15_na15 | 0.12 | 0.09 |

ExternalSignal_nr1_na1 | 0.29 | 0.20 |

ExternalSignal_nr5_na5 | 0.39 | 0.28 |

ExternalSignal_nr10_na10 | 0.09 | 0.06 |

**Table 2.**Pearson correlation coefficient $\rho $ between input attention and true interaction matrix.

Network | $\mathit{\rho}$ |
---|---|

FullyConnected | −0.018 |

FullyRepressed | 0.0 |

MasterRegulator | −0.080 |

SparseConnection | −0.10 |

mediumConnection | −0.0087 |

Oscillating_nr1_na1 | −0.0070 |

Oscillating_nr5_na5 | 0.017 |

Oscillating_nr10_na10 | 0.061 |

Oscillating_nr15_na15 | 0.12 |

ExternalSignal_nr1_na1 | −0.0020 |

ExternalSignal_nr5_na5 | 0.075 |

ExternalSignal_nr10_na10 | 0.021 |

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**MDPI and ACS Style**

Monti, M.; Fiorentino, J.; Milanetti, E.; Gosti, G.; Tartaglia, G.G.
Prediction of Time Series Gene Expression and Structural Analysis of Gene Regulatory Networks Using Recurrent Neural Networks. *Entropy* **2022**, *24*, 141.
https://doi.org/10.3390/e24020141

**AMA Style**

Monti M, Fiorentino J, Milanetti E, Gosti G, Tartaglia GG.
Prediction of Time Series Gene Expression and Structural Analysis of Gene Regulatory Networks Using Recurrent Neural Networks. *Entropy*. 2022; 24(2):141.
https://doi.org/10.3390/e24020141

**Chicago/Turabian Style**

Monti, Michele, Jonathan Fiorentino, Edoardo Milanetti, Giorgio Gosti, and Gian Gaetano Tartaglia.
2022. "Prediction of Time Series Gene Expression and Structural Analysis of Gene Regulatory Networks Using Recurrent Neural Networks" *Entropy* 24, no. 2: 141.
https://doi.org/10.3390/e24020141