Distant regulatory sequences affect transcription through long-range chromatin interactions. Visualization of transcriptional activity of genes that compete for distant elements, using the globin locus as a model, has revealed the dynamics of chromatin interactions in vivo. Multiple genes appear to be transcribed alternately rather than at the same time to generate several messenger RNAs in one cell. The regulator may stably complex with one gene at a time and switch back and forth between genes in a flip-flop mechanism.

Locus control regions (LCRs) are responsible for initiating and maintaining a stable tissue-specific open chromatin structure of a locus. In transgenic mice, LCRs confer high level expression on linked genes independent of position in the mouse genome. Here we show that an incomplete LCR loses this property when integrated into heterochromatic regions. Two disruption mechanisms were observed. One is classical position-effect variegation, resulting in continuous transcription in a clonal subpopulation of cells. The other is a novel mechanism resulting in intermittent gene transcription in all cells. We conclude that only a complete LCR fully overcomes heterochromatin silencing and that it controls the level of transcription by ensuring activity in all cells at all times rather than directly controlling the rate of transcription.

The hedgehog signaling pathway organizes the developing ventral neural tube by establishing distinct neural progenitor fates along the dorsoventral axis. Smoothened (Smo) is essential for all Hedgehog (Hh) signaling, and genetic inactivation of Smo cells autonomously blocks the ability of cells to transduce the Hh signal. Using a chimeric approach, we examined the behavior of Smo null mutant neural progenitor cells in the developing vertebrate spinal cord, and we show that direct Hh signaling is essential for the specification of all ventral progenitor populations. Further, Hh signaling extends into the dorsal half of the spinal cord including the intermediate Dbx expression domain. Surprisingly, in the absence of Sonic hedgehog (Shh), we observe the presence of a Smo-dependent Hh signaling activity operating in the ventral half of the spinal cord that most likely reflects Indian hedgehog (Ihh) signaling originating from the underlying gut endoderm. Comparative studies of Shh, Smo, and Gli3 single and compound mutants reveal that Hh signaling acts in part to specify neural cell identity by counteracting the repressive action of Gli3 on p0, p1, p2, and pMN formation. However, whereas these cell identities are restored in Gli3/Smo compound mutants, correct stratification of the rescued ventral cell types is lost. Thus, Hh signaling is essential for organizing ventral cell pattern, possibly through the control of differential cell affinities.

We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human 13-globin genes in compound EKLF knockout/human I~-locus transgenic mice. EKLF affects only the adult mouse 13-globin genes in homozygous knockout mice; heterozygous mice are unaffected. Here we show that EKLF knockout mice express the human e and 7-globin genes normally in embryonic red cells. However, fetal liver erythropoiesis, which is marked by a period of 7" and 13-gene competition in which the genes are alternately transcribed, exhibits an altered ratio of 7" to 13-gene transcription. EKLF heterozygous fetal livers display a decrease in the number of transcriptionally active 13 genes with a reciprocal increase in the number of transcriptionally active 7 genes. 13-gene transcription is absent in homozygous knockout fetuses with coincident changes in chromatin structure at the 13 promoter. There is a further increase in the number of transcriptionally active 7 genes and accompanying 7 gene promoter chromatin alterations. These results indicate that EKLF plays a major role in 7-and I~-gene competition and suggest that EKLF is important in stabilizing the interaction between the Locus Control Region and the 13-globin gene. In addition, these findings provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by altering the frequency and/or duration of transcriptional periods of a gene rather than changing the rate of transcription.