We have demonstrated that the DAR-FFC in which the two repressor regulators are both autoregulated is a distinct and significant network motif in the human transcriptional regulatory network. We were able to improve the resolution power for analyzing the network circuitry by devising an autoregulation-based FFC classification and combining it with the mode-based FFC classification. By focusing on the autoregulation-integrated FFC, we demonstrated the analytical relevance of such small-scale integrated circuits that are positioned between elementary circuit units (e.g. autoregulation and simple FFCs) and large-scale integrated circuits (e.g. 'dense overlapping regulon') . This autoregulation-based classification has an important logical feature in that it merely depends on the circuit structure without introducing any qualitative features (i.e. repressor or activator) of the regulator, and it still enabled the delineation of specific antimotifs.
We also showed that the overall FFC, coherent FFC and coherent type-1 FFC appearances were each significantly overrepresented in the human transcriptional regulatory network based on the conventional mode-based classification. These results are consistent with previous findings in E. coli and yeast, suggesting strong evolutionary conservation of the usage of FFCs, particularly type-1 coherent FFCs, at the system level.
The autoregulation-and-mode combined classification enabled us to identify two antimotifs that were both composed of an originating repressor regulator with autoregulation and an intermediary regulator without autoregulation irrespective of the mode. This autoregulation configuration is likely to have a specific disadvantage for feedforward synergistic control of the effecter. From the viewpoint of circuit dynamics, negative autoregulation formed by a repressor has been reported to speed up the response of its own regulator . Therefore, the aforementioned antimotifs that each form a circuit of a quick originating regulator aided by a slower modifier (i.e. intermediary regulator) seem to be inappropriate for fine-tuning of the target gene, possibly because the modifier is required to monitor the originating regulator at a higher frequency than the switching frequency of the originating regulator.
The combined classification further revealed the DAR-FFC that targets effecter genes as a novel network motif that comprises two repressors with autoregulation. The overrepresentation of the number of DAR-FFC appearances is possibly explained by the facts that the autorepression integrated in the DAR-FFC provides robustness against stochastic perturbation  and accelerates reaching a stable transcription level , which consequently make DAR-FFCs advantageous for controlling the effecters. This notion is specifically supported by the finding that the most preferred molecular function of the DAR-FFC-targeted effecters was 'growth factor activity' (Table 2), since cellular responses to a growth factor have been reported to require a set of key 'repressive' transcriptional regulators to achieve tightly controlled signal attenuation processes . Consequently, the swiftness and robustness of DAR-FFCs are suitable properties for the signaling systems of various growth factors.
In addition to 'growth factor activity', the effecter functions targeted by the DAR-FFCs demonstrated a marked preference for intercellular communications through humoral, neuronal and ECM-mediated signaling modalities, representing the major intercellular communications in higher eukaryotes. Another functionality significantly preferred by the DAR-FFC-targeted effecters included transcription-modifying activities that increase the information integration capacities at the transcriptional level. These properties of DAR-FFCs have a crucial advantage for enhancing the information transmission and integration that is inevitable for the inflated needs of information processing that are possibly brought about multicellularization.
Notably, and consistent with the above notion, we found that 'multicellular organismal development' was by far the most preferred biological process among DAR-FFC-targeted effecters. It is also noteworthy that 'multicellular organismal development' was followed in the list by biological processes related to the neuronal cell fate program, namely 'nervous system development', 'Wnt receptor signaling pathway' and 'axon guidance'. This preference for nervous system development is a general feature of DAR-FFCs because all the individual DAR-FFC connections identified in the present study had effecter genes with preferred functions within 'multicellular organismal development' and 'nervous system development'. Furthermore, our network analyses revealed that the DAR-FFCs were densely interconnected, and that individual DAR-FFCs formed a higher-order DAR-FFC topology. This topology included a possible 'higher-order originating regulator hub' that potentially spreads regulatory effects through DAR-FFC-mediated transmissions. These dense interlinking features of DAR-FFCs are likely to provide further robustness for transcriptional regulatory networks involved in multicellularity and nervous system development.
Based on the results of the network motif representation analyses, GO functional analyses and higher-order network structures, we suggest that the DAR-FFC identified in the present study is a distinctive integrated network motif endowed with properties that are indispensable for forming the transcriptional regulatory circuits essential for multicellular organization and nervous system development. An intelligible and comprehensive description of the transcriptional regulatory system requires an appropriate set of network motifs that would include integrated network motifs such as the DAR-FFC. It is necessary to elucidate other potential integrated network motifs with a sufficient descriptive power for understanding the gene regulatory system. The degree of integration or abstraction of these motifs would vary depending on the purpose of the description of the system.