Virus-assisted mapping of neural inputs to a feeding center in the hypothalamus.

We report the development of a pseudorabies virus that can be used for retrograde tracing from selected neurons. This virus encodes a green fluorescent protein marker and replicates only in neurons that express the Cre recombinase and in neurons in synaptic contact with the originally infected cells. The virus was injected into the arcuate nucleus of mice that express Cre only in those neurons that express neuropeptide Y or the leptin receptor. Sectioning of the brains revealed that these neurons receive inputs from neurons in other regions of the hypothalamus, as well as the amygdala, cortex, and other brain regions. These data suggest that higher cortical centers modulate leptin signaling in the hypothalamus. This method of neural tracing may prove useful in studies of other complex neural circuits.

The embryonic transcription factor stage specific activator protein contains a potent bipartite activation domain that interacts with several RNA polymerase II basal transcription factors.

Stage specific activator protein (SSAP) is a member of a newly discovered class of transcription factors that contain motifs more commonly found in RNA-binding proteins. Previously, we have shown that SSAP specifically binds to its recognition sequence in both the double strand and the single strand form and that this DNA-binding activity is localized to the N-terminal RNA recognition motif domain. Three copies of this recognition sequence constitute an enhancer element that is directly responsible for directing the transcriptional activation of the sea urchin late histone H1 gene at the midblastula stage of embryogenesis. Here we show that the remainder of the SSAP polypeptide constitutes an extremely potent bipartite transcription activation domain that can function in a variety of mammalian cell lines. This activity is as much as 3 to 5 times stronger than VP16 at activating transcription and requires a large stretch of amino acids that contain glutamine-glycine rich and serine-threonine-basic amino acid rich regions. We present evidence that SSAP's activation domain shares targets that are also necessary for activation by E1a and VP16. Finally, SSAP's activation domain is found to participate in specific interactions in vitro with the basal transcription factors TATA-binding protein, TFIIB, TFIIF74, and dTAF(II) 110.

The embryonic enhancer-binding protein SSAP contains a novel DNA-binding domain which has homology to several RNA-binding proteins.

Stage-specific activator protein (SSAP) is a 43-kDa polypeptide that binds to an enhancer element of the sea urchin late histone H1 gene. This enhancer element mediates the transcriptional activation of the late histone H1 gene in a temporally specific manner at the mid-blastula stage of embryogenesis. We have cloned cDNAs encoding SSAP by using polyclonal antibodies raised against purified SSAP to screen expression libraries. SSAP is unrelated to previously characterized transcription factors; however, it exhibits striking homology to a large family of proteins involved in RNA processing. The protein is a sequence-specific DNA-binding protein that recognizes both single- and double-stranded DNA. The DNA-binding domain of the protein was localized to the conserved RNA recognition motif (RRM). In addition to tandem copies of this conserved domain, SSAP contains a central domain that is rich in glutamine and glycine and a C-terminal domain that is enriched in serine, threonine, and basic amino acids. Overexpression of SSAP in sea urchin embryos by microinjection of either synthetic mRNA or an SSAP expression vector results in four- to eightfold transactivation of target reporter genes that contain the enhancer sequence. Transactivation occurs beginning only at the mid-blastula stage of development, suggesting that SSAP must be modified in a stage-specific manner in order to activate transcription. In addition, there are a number of other RRM-containing proteins that contain glutamine-rich regions which are postulated to function in the regulation of RNA processing. Instead, we suggest that SSAP is a member of a family of glutamine-rich RRM proteins which constitute a novel class of transcription factors.

Purification and characterization of the stage-specific embryonic enhancer-binding protein SSAP-1.

We have demonstrated that a highly conserved segment of DNA between positions -288 and -317 (upstream sequence element IV [USE IV]) is largely responsible for the transcriptional activation of the sea urchin H1-beta histone gene during the blastula stage of embryogenesis. This sequence is capable of acting as an embryonic enhancer element, activating target genes in a stage-specific manner. Nuclear extracts prepared from developmentally-staged organisms before and after the gene is activated all contain a factor which specifically binds to the enhancer. We have purified a 43-kDa polypeptide which binds to and footprints the USE IV enhancer element. We refer to this protein as stage-specific activator protein 1 (SSAP-1). Early in development before the enhancer is active, SSAP appears as a 43-kDa monomer, but it undergoes a change in its molecular weight beginning at about 12 h postfertilization (early blastula) which precisely parallels the increase in H1-beta gene expression. Modified SSAP has an apparent molecular mass of approximately 90 to 100 kDa and contains at least one 43-kDa SSAP polypeptide. Thus, it is the disappearance of the 43-kDa species and the appearance of the 90- to 100-kDa species which coincide with the H1-beta gene activation. The correlation between the change in molecular weight of SSAP and the stage-specific activation of H1-beta gene expression strongly suggests that this higher-molecular-weight form of SSAP is directly responsible for the blastula stage-specific transcriptional activation of the late H1 gene.