Chromatin environments differ greatly within a eukaryotic genome, depending on expression state, chromosomal location, and nuclear position. In genomic regions characterized by high repeat content and high gene density, chromatin structure must silence transposable elements but permit expression of embedded genes. We have investigated one such region, chromosome 4 of Drosophila melanogaster. Using chromatin-immunoprecipitation followed by microarray (ChIP–chip) analysis, we examined enrichment patterns of 20 histone modifications and 25 chromosomal proteins in S2 and BG3 cells, as well as the changes in several marks resulting from mutations in key proteins. Active genes on chromosome 4 are distinct from those in euchromatin or pericentric heterochromatin: while there is a depletion of silencing marks at the transcription start sites (TSSs), HP1a and H3K9me3, but not H3K9me2, are enriched strongly over gene bodies. Intriguingly, genes on chromosome 4 are less frequently associated with paused polymerase. However, when the chromatin is altered by depleting HP1a or POF, the RNA pol II enrichment patterns of many chromosome 4 genes shift, showing a significant decrease over gene bodies but not at TSSs, accompanied by lower expression of those genes. Chromosome 4 genes have a low incidence of TRL/GAGA factor binding sites and a low Tm downstream of the TSS, characteristics that could contribute to a low incidence of RNA polymerase pausing. Our data also indicate that EGG and POF jointly regulate H3K9 methylation and promote HP1a binding over gene bodies, while HP1a targeting and H3K9 methylation are maintained at the repeats by an independent mechanism. The HP1a-enriched, POF-associated chromatin structure over the gene bodies may represent one type of adaptation for genes embedded in repetitive DNA.

Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27(Kip1) having the broadest expression, and both p57(Kip2) and p21(Cip1) showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57(Kip2);p27(Kip1) double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity of Cyclin D1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57(Kip2) in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.

Polycomb bodies are foci of Polycomb proteins in which different Polycomb target genes are thought to co-localize in the nucleus, looping out from their chromosomal context. We have shown previously that insulators, not Polycomb response elements (PREs), mediate associations among Polycomb Group (PcG) targets to form Polycomb bodies. Here we use live imaging and 3C interactions to show that transgenes containing PREs and endogenous PcG-regulated genes are targeted by insulator proteins to different nuclear structures depending on their state of activity. When two genes are repressed, they co-localize in Polycomb bodies. When both are active, they are targeted to transcription factories in a fashion dependent on Trithorax and enhancer specificity as well as the insulator protein CTCF. In the absence of CTCF, assembly of Polycomb bodies is essentially reduced to those representing genomic clusters of Polycomb target genes. The critical role of Trithorax suggests that stable association with a specialized transcription factory underlies the cellular memory of the active state.

Proper central nervous system (CNS) function depends critically on the generation of functionally distinct neuronal types in specific and reproducible positions. The generation of neuronal diversity during CNS development involves a fine balance between dividing neural progenitors and the differentiated neuronal progeny that they produce. However, the molecular mechanisms that regulate these processes are still poorly understood. Here, we show that the Prox1 transcription factor, which is expressed transiently and specifically in spinal interneurons, plays an important role in neurogenesis. Using both gain-and loss-offunction approaches, we find that Prox1 is capable of driving neuronal precursors out of the cell cycle and can initiate limited expression of neuronal proteins. Using RNAi approaches, we show that Prox1 function is required to execute a neurogenic differentiation program downstream of Mash1 and Ngn2. Our studies demonstrate an important, spinal interneuron-specific role for Prox1 in controlling steps required for both cell-cycle withdrawal and differentiation. Developmental Dynamics 237:393-402, 2008.

Ataxin-1 (ATXN1), a causative factor for spinocerebellar ataxia type 1 (SCA1), and the related Brother of ATXN1 (BOAT1) are human proteins involved in transcriptional repression. So far, little is known about which transcriptional pathways mediate the effects of ATXN1 and BOAT1. From our analyses of the properties of BOAT1 in Drosophila and of both proteins in mammalian cells, we report here that BOAT1 and ATXN1 are components of the Notch signalling pathway. In Drosophila, BOAT1 compromises the activities of Notch. In mammalian cells, both ATXN1 and BOAT1 bind to the promoter region of Hey1 and inhibit the transcriptional output of Notch through direct interactions with CBF1, a transcription factor that is crucial for the Notch pathway. Our results suggest that, in addition to their involvement in SCA1, ATXN1 and BOAT1 might participate in several Notch-controlled developmental and pathological processes.