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Holoprosencephaly (HPE) is a genetically and phenotypically heterogenous disorder involving the development of forebrain and midface, with an incidence of 1:16,000 live born and 1:250 induced abortions. This disorder is associated with several distinct facies and phenotypic variability: in the most extreme cases, anophthalmia or cyclopia is evident along with a congenital absence of the mature nose. The less severe form features facial dysmorphia characterized by ocular hypertelorism, defects of the upper lip and/or nose, and absence of the olfactory nerves or corpus callosum. Several intermediate phenotypes involving both the brain and face have been described. One of the gene loci, HPE3, maps to the terminal band of chromosome 7. We have performed extensive physical mapping studies and established a critical interval for HPE3, and subsequently identified the sonic hedgehog (SHH) gene as the prime candidate for the disorder. SHH lies within 15-250 kilobases (kb) of chromosomal rearrangements associated with HPE, suggesting that a 'position effect' has an important role in the aetiology of HPE. As detailed in the accompanying report, this role for SHH is confirmed by the detection of point mutations in hereditary HPE patients.

Fluorescence in situ hybridization to metaphase chromosomes or chromatin fibers In Interphase nuclei is a powerful technique in mapping genes and DNA segments to specific chromosome region. We have been able to release the chromatin fibers from cells arrested at GI and G2 phases using different drugs and a simple alkailne lysis procedure. We have also demonstrated specific hybridization of fluorescencelabeled probes to single-copy genoic DNA sequences on the free chromatins. Fluorescence in situ hybridztion signals have been detected for sequences separated as close as 21 klobase pairs and as far as 350 kilobase pairs, with excellent correspondence between the observed and expetd distances. The resolution of this technique should approach 10 kilobase pairs and its coverage should span millions of base pairs. Therefore, free chromatin mapping can be generally used to study the structure and organization of mammalian genomes.

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Histone acetylation plays an important role in regulating chromatin structure and thus gene expression. Here we describe the functional characterization of HDAC4, a human histone deacetylase whose C-terminal part displays significant sequence similarity to the deacetylase domain of yeast HDA1. HDAC4 is expressed in various adult human tissues, and its gene is located at chromosome band 2q37. HDAC4 possesses histone deacetylase activity intrinsic to its C-terminal domain. When tethered to a promoter, HDAC4 represses transcription through two independent repression domains, with repression domain 1 consisting of the N-terminal 208 residues and repression domain 2 containing the deacetylase domain. Through a small region located at its N-terminal domain, HDAC4 interacts with the MADS-box transcription factor MEF2C. Furthermore, HDAC4 and MEF2C individually upregulate but together downmodulate c-jun promoter activity. These results suggest that HDAC4 interacts with transcription factors such as MEF2C to negatively regulate gene expression.In eukaryotic cells, genetic information is packaged into chromatin, a highly organized DNA-protein complex which controls gene activities. A central question in studying eukaryotic gene regulation is how the generally repressive chromatin structure is regulated when necessary. In the past several years, three regulatory mechanisms have been recognized: DNA methylation, posttranslational modifications of histones, and ATP-dependent chromatin remodeling (53,55,57). The most extensively studied form of posttranslational modifications of histones is acetylation of ε-amino groups of lysine residues located at the flexible N-terminal tails of core histones (53, 55). The level of histone acetylation at a given region of chromatin correlates well with its transcriptional activity (39). Mechanistically, histone acetylation affects nucleosome stability and/or internucleosomal interaction (2, 29). The dynamic level of histone acetylation in vivo is maintained through opposing actions of histone acetyltransferases and deacetylases. Several known transcriptional coactivators possess intrinsic histone acetyltransferase activity (14,27,49,57).The first histone deacetylase, originally called HD1 (histone deacetylase 1) and later renamed HDAC1 (histone deacetylase 1), was cloned from mammalian cells (18, 50). HDAC1 was found to be highly homologous to the known yeast transcriptional coregulator RPD3 (50). Two HDAC1 homologs (HDAC2 and HDAC3) have been cloned from human cDNA libraries (10,58,59). Transcriptional repressors recruit RPD3 or HDAC1 to -3 to downregulate transcription (reviewed in references 41 and 56). The deacetylase activity of HDAC1 and RPD3 has been found to be important for transcriptional repression (18, 24), suggesting that histone deacetylation directly leads to transcriptional repression. Consistent with this contention, recruitment of RPD3 by the yeast repressor Ume6 leads to local histone deacetylation and formation of a highly localized domain of repressed chromatin in vivo...