Drosophila sine oculis and eyes absent genes synergize in compound-eye formation. The murine homologues of these genes, Six and Eya, respectively, show overlapping expression patterns during development. We hypothesized that Six and Eya proteins cooperate to regulate their target genes. Cotransfection assays were performed with various combinations of Six and Eya to assess their effects on a potential natural target, myogenin promoter, and on a synthetic promoter, the thymidine kinase gene promoter fused to multimerized Six4 binding sites. A clear synergistic activation of these promoters was observed in certain combinations of Six and Eya. To investigate the molecular basis for the cooperation, we first examined the intracellular distribution of Six and Eya proteins in transfected COS7 cells. Coexpression of Six2, Six4, or Six5 induced nuclear translocation of Eya1, Eya2, and Eya3, which were otherwise distributed in the cytoplasm. In contrast, coexpression of Six3 did not result in nuclear localization of any Eya proteins. Six and Eya proteins were coimmunoprecipitated from nuclear extracts prepared from cotransfected COS7 cells and from rat liver. Six domain and homeodomain, two evolutionarily conserved domains among various Six proteins, were necessary and sufficient for the nuclear translocation of Eya. In contrast, the Eya domain, a conserved domain among Eya proteins, was not sufficient for the translocation. A specific interaction between the Six domain and homeodomain of Six4 and Eya2 was observed by yeast two-hybrid analysis. Our results suggest that transcription regulation of certain target genes by Six proteins requires cooperative interaction with Eya proteins: complex formation through direct interaction and nuclear translocation of Eya proteins. This implies that the synergistic action of Six and Eya is conserved in the mouse and is mediated through cooperative activation of their target genes.Six genes are mouse homologues of the Drosophila sine oculis (so) gene, which is essential for compound-eye formation (9, 31). Six members of the Six family of genes have so far been identified in the mouse (17,18,27,28,35). Each Six gene shows a specific expression pattern during development of the mouse embryo. Six1 and Six2 show expression in mesenchymal cells around E8.5 to E10.5 and in muscles and limb tendons in later stages (28). Six3 is expressed in the rostral forebrain in earlier stages and is confined to the prospective eye region (27). Six4 proteins are distributed in the peripheral region of the mantle layer of the developing brain and spinal cord and in various ganglia between E9.5 and E14.5 (25). Six5 mRNA is expressed as early as E7 and is abundantly expressed in neonatal heart and skeletal muscles (24). Human SIX5 resides downstream of a CTG repeat, whose expansion leads to myotonic dystrophy (DM) (7). Since SIX5 is expressed in several tissues affected by DM and the transcription of SIX5 is repressed by the causative a CTG repeat expansion, it has been proposed that SIX5 is involved in som...

A similar gene network was found to control chick myogenesis, in which Six1, Eya2 and Dach2 synergistically regulate the expression of myogenic genes such as myogenin and MyoD (Heanue et Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wildtype otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1-and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.

Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity. To date, no method for full-length total RNA sequencing of single cells has been developed despite the potential of this technology for single-cell biology. Here we describe random displacement amplification sequencing (RamDA-seq), the first full-length total RNA-sequencing method for single cells. Compared with other methods, RamDA-seq shows high sensitivity to non-poly(A) RNA and near-complete full-length transcript coverage. Using RamDA-seq with differentiation time course samples of mouse embryonic stem cells, we reveal hundreds of dynamically regulated non-poly(A) transcripts, including histone transcripts and long noncoding RNA Neat1. Moreover, RamDA-seq profiles recursive splicing in >300-kb introns. RamDA-seq also detects enhancer RNAs and their cell type-specific activity in single cells. Taken together, we demonstrate that RamDA-seq could help investigate the dynamics of gene expression, RNA-processing events and transcriptional regulation in single cells.

The members of the Six gene family were identified as homologues of Drosophila sine oculis which is essential for compound-eye formation. The Six proteins are characterized by the Six domain and the Six-type homeodomain, both of which are essential for specific DNA binding and for cooperative interactions with Eya proteins. Mammals possess six Six genes which can be subdivided into three subclasses, and mutations of Six genes have been identified in human genetic disorders. Characterization of Six genes from various animal phyla revealed the antiquity of this gene family and roles of its members in several different developmental contexts. Some members retain conserved roles as components of the Pax-Six-Eya-Dach regulatory network, which may have been established in the common ancestor of all bilaterians as a toolbox controlling cell proliferation and cell movement during embryogenesis. Gene duplications and cis-regulatory changes may have provided a basis for diverse functions of Six genes in different animal lineages.

Many aspects of behavior and physiology, including sleep/ awake cycles and hormone levels, keep a rhythm with about a 24-h period, even under constant conditions without any external time cues (1). Circadian rhythms are generated by a self-sustaining time-measuring system called the circadian clock. In mammals, the hypothalamic suprachiasmatic nucleus (SCN) functions as the master clock, and circadian clocks are also located in peripheral tissues such as the liver (2-5). In individual cells, clock genes and their products form transcriptional/translational feedback loops (6). The basic helix-loop-helix (bHLH)-PAS transcription factors CLOCK and BMAL1 play a role as positive factors in the loops, and the heterodimer of these proteins binds to the CACGTG E-box or related E-box-like sequences to transactivate a wide range of target genes, including Per and Cry (7-10). Translated PER and CRY proteins then bind to the CLOCK-BMAL1 complex, leading to the suppression of E-box-dependent transactivation. This negative-feedback mechanism forms a molecular clock generating circadian rhythms. In addition to the Ebox element, the D-box element and the REV-ERB/ROR-binding element (RRE) form a regulatory network of gene expression, governing coordinately circadian transcriptional oscillations (11,12). The D-box element is activated and repressed by DBP and E4BP4, respectively, while RRE is activated and repressed by RORs and REV-ERBs, respectively.During the circadian cycling of the transcriptional/translational steps, posttranslational modifications, such as phosphorylation, regulate the clock proteins, in terms of activity, stability, localization, and interaction (13). It was reported previously that CLOCK and BMAL1 are phosphorylated in a time-of-day-dependent manner (14)(15)(16)(17). CLOCK phosphorylation at its DNA-binding domain (16, 18) may be important for rhythmic inhibition of the ability of the CLOCK-BMAL1 complex to bind to the E-box element. This is consistent with the observation that the CLOCK-BMAL1 complex rhythmically dissociates from the E-box in the locus of the Dbp gene (19). Here we found in vivo binding sites of CLOCK protein in the mouse liver in a genome-wide manner by chromatin immunoprecipitation-sequencing (ChIP-Seq) analysis. Previous ChIP-Seq studies of circadian clocks confirmed CLOCK-BMAL1 binding to canonical motifs instead of finding all potential binding motifs (20)(21)(22)(23). In this study, significant CLOCK-binding motifs were comprehensively examined by developing a bioinformatics method, MOCCS (motif centrality analysis of ChIP-Seq), which analyzes the frequency distribution of DNA sequences centered at DNA-binding sites found by ChIPSeq analyses. In parallel, all the rhythmic transcripts in the liver were identified by circadian deep-sequencing analysis of poly(A)-tailed RNA and small RNA. Based on these data, we demonstrate the functional importance of rhythmic posttranscriptional regulations, such as microRNA (miRNA)-mediated gene silencing, in dynamic circadian RNA rhythms.