CTCF-dependent enhancer blockers at the upstream region of the chicken alpha-globin gene domain

The eukaryotic genome is partitioned into chromatin domains containing coding and intergenic regions. Insulators have been suggested to play a role in establishing and maintaining chromatin domains. Here we describe the identification and characterization of two separable enhancer blocking elements located in the 5′ flanking region of the chicken α-globin domain, 11–16 kb upstream of the embryonic α-type π gene in a DNA fragment harboring a MAR (matrix attachment region) element and three DNase I hypersensitive sites (HSs). The most upstream enhancer blocking element co-localizes with the MAR element and an erythroid-specific HS. The second enhancer blocking element roughly co-localizes with a constitutive HS. The third erythroid-specific HS present within the DNA fragment studied harbors a silencing, but not an enhancer blocking, activity. The 11 zinc-finger CCCTC-binding factor (CTCF), which plays an essential role in enhancer blocking activity in many previously characterized vertebrate insulators, is found to bind the two α-globin enhancer blocking elements. Detailed analysis has demonstrated that mutation of the CTCF binding site within the most upstream enhancer blocking element abolishes the enhancer blocking activity. The results are discussed with respect to special features of the tissue-specific α-globin gene domain located in a permanently open chromatin area.     

Detection of a new tissue-specific enhancer in the ct6 region of the regulatory region of the drosophila cut locus]

The cut locus of Drosophila is an interesting example of a complex eukaryotic locus responsible for the development of many tissues and organs. Most of this locus is regulatory. The entire locus was cloned by Tchurikov et al. in 1986 and Blochlinger et al. in 1988. The wing ctn enhancer located 80 kb upstream of the promoter was earlier found in a 2.7 kb EcoRI-BamHI DNA fragment. The locus region 65-80 kb remote from the promoter was assumed to control the development of wings and vibrissae. We have found a new enhancer region in the ct6 region of the locus, which was in a 5 kb BamHI-EcoRI DNA fragment adjacent to the ctn enhancer. This region is responsible for the expression of the reporter lacZ gene in many tissues and organs at all stages of Drosophila development (at least in the intestine, Malpighian tubules, thoracic and abdominal sensory organs, thoracic ganglia and in ring glands). Thus, the region located 75 kb upstream of the promoter has some properties of the locus control region (LCR).     

New genes interacting with the zeste gene in Drosophila melanogaster]

We have found that mutations in the enhancer of yellow, 1,2 and 3 loci strongly enhance the effect of zv77h-mutation (full inactivation of the zeste locus) on the white locus expression. Their effect is realized through the distal white enhancer which is located 1,1 kb upstream to the promoter. It is suggested that the protein products of enhancers of yellow 1,2 and 3 represent a family of proteins which, like zeste protein, are responsible for formation of contacts between elements located at a large distance in the genome.     

人乳头瘤病毒6b型(HPV-6b)的上游调控区在大肠杆菌中也具有增强子样的效应

采用痘苗病毒7.5k、11k及N2启动子,以大肠杆菌β-半乳糖苷酶(β-lac)、氯霉素乙酰基转移酶(CAT)作为标记基因,构建了大肠杆菌增强子样序列的系列检测载体。采用上述检测载体发现人乳头瘤病毒6b(Human papillomavirus type 6b,HPV-6b)上游调控区(Upstream regulating region,URR)中540bp的Sau3A-Nar Ⅰ片段,在大肠杆菌中对痘苗病毒启动子控制的CAT基因的表达有明显的增强作用,可使基因表达提高4～5倍;对β-lac基因的表达可提高3～6倍。根据这一片段的插入方向增强基因表达水平有所不同。     

Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific.

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.