SUMMARY Combinatorial interactions among transcription factors are critical to directing tissue-specific gene expression. To build a global atlas of these combinations, we have screened for physical interactions among the majority of human and mouse DNA-binding transcription factors (TFs). The complete networks contain 762 human and 877 mouse interactions. Analysis of the networks reveals that highly connected TFs are broadly expressed across tissues, and that roughly half of the measured interactions are conserved between mouse and human. The data highlight the importance of TF combinations for determining cell fate, and they lead to the identification of a SMAD3/FLI1 complex expressed during development of immunity. The availability of large TF combinatorial networks in both human and mouse will provide many opportunities to study gene regulation, tissue differentiation, and mammalian evolution.

Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites, we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process.

Foxp3 plays an important role in the development and the function of regulatory T cells (Treg). Both the induction and maintenance of Foxp3 gene expression are controlled by several regulatory regions including two enhancers in the conserved noncoding sequences (CNS). The functions of Enhancer 1 in CNS1 are well established, whereas those of Enhancer 2 in CNS2 remain unclear. Although CNS2 contains enhancer activity, methylated CpG sequences in this region prevent Foxp3 gene expression in Foxp3− T cells. These sequences are however demethylated in Foxp3+ Treg by mechanisms as yet unknown. To investigate the role of CNS2, we have determined the Enhancer 2 core sequence by luciferase reporter assays in the absence of methylation to exclude the inhibitory effect, and shown that transcription factors AP-1, Stat5 and Creb cooperate in regulating Enhancer 2 activity. We have then determined the methylation sensitivity of each of the transcription factors. AP-1 was found to be methylation sensitive as has previously been described for Creb. However, Stat5 was active even when its binding site in CNS2 was methylated. Stat5 binding to Enhancer 2 occurred early, and preceded that of AP-1 and Creb during Treg induction. In addition, Stat5 activation is itself dependent on TGF-β signaling through Smad3 mediated blockade of Socs3 expression. These findings suggest that Stat5 is a key regulator for opening up the CNS2 region during iTreg induction, while AP-1 and Creb maintain Enhancer 2 activity.

The survival of motor neuron (SMN) protein, responsible for the neurodegenerative disease spinal muscular atrophy (SMA), oligomerizes and forms a stable complex with seven other major components, the Gemin proteins. Besides the SMN protein, Gemin2 is a core protein that is essential for the formation of the SMN complex, although the mechanism by which it drives formation is unclear. We have found a novel interaction, a Gemin2 self-association, using the mammalian two-hybrid system and the in vitro pull-down assays. Using in vitro dissociation assays, we also found that the self-interaction of the amino-terminal SMN protein, which was confirmed in this study, became stable in the presence of Gemin2. In addition, Gemin2 knockdown using small interference RNA treatment revealed a drastic decrease in SMN oligomer formation and in the assembly activity of spliceosomal small nuclear ribonucleoprotein (snRNP). Taken together, these results indicate that Gemin2 plays an important role in snRNP assembly through the stabilization of the SMN oligomer/complex via novel self-interaction. Applying the results/techniques to amino-terminal SMN missense mutants that were recently identified from SMA patients, we successfully showed that amino-terminal self-association, Gemin2 binding, the stabilization effect of Gemin2, and snRNP assembly activity were all lowered in the mutant SMN(D44V), suggesting that instability of the amino-terminal SMN self-association may cause SMA in patients carrying this allele. Spinal muscular atrophy (SMA)2 is a common autosomal recessive disease that is clinically classified into three types, I-III, based on the severity of motor neuron degeneration and the age of onset (1-3). Two nearly identical copies of the survival of motor neuron genes (SMN1 and SMN2) are located on the human chromosome, 5q13, whereas other eukaryotic species have only one copy of the SMN gene. Homozygous deletions of, or mutations in the SMN1 gene are responsible for SMA (4). SMN is expressed ubiquitously and is a core component of a self-assembling multiprotein complex, the SMN complex, consisting of SMN and Gemin proteins, which plays an essential role in the assembly of the spliceosomal small nuclear ribonucleoproteins (snRNPs) and in pre-mRNA splicing (5, 6).Most of the missense mutations in SMA patients are located in exon 6 of SMN1; whereas nonsense and frameshift mutations are widely spread throughout the entire gene (7). Exon 6 of SMN1 encodes a self-association domain, and deletions or mutations of this domain result in a decrease in oligomer formation of SMN, which correlates with the severity of SMA (8). SMN oligomerization is also a prerequisite for high affinity binding of the SMN complex to spliceosomal snRNPs (9). The self-association domains of SMN were identified by surface plasmon resonance analysis; both the carboxyl-terminal exon 6-encoded region and the amino-terminal exon 2b-encoded region contributed to self-association (10). This suggests a mechanism in which the self-association formation involves eit...