Biomedical subjects
Publications and source records attributed to Gary Felsenfeld.
Chromatin is the complex of DNA and proteins in which the genetic material is packaged inside the cells of organisms with nuclei. Chromatin structure is dynamic and exerts profound control over gene expression and other fundamental cellular processes. Changes in its structure can be inherited by the next generation, independent of the DNA sequence itself.
During the past several years there has been intense interest in the mechanisms by which gene expression is regulated within the eukaryotic nucleus. We have made use of an avian beta-globin locus to study various aspects of this problem, some of which are amenable to quantitative analysis. In the course of this work we have identified the transcription factor GATA-1, which is an essential regulatory factor for virtually all erythroid-specific genes, and studied the structure of its complex with its specific DNA binding site. The way in which GATA-1 forms tight interactions with DNA led to an understanding of how other zinc finger proteins of this class bind to DNA. We have extended such studies to examine interactions with DNA packaged as chromatin, and to studies of chromatin structure and function at higher levels of organization.
The anion exchanger protein 1 (AE1; band 3) is an abundant erythrocyte transmembrane protein that regulates chloride-bicarbonate exchange and provides an attachment site for the erythrocyte membrane skeleton on the cytoplasmic domain. We analyzed the function of the erythroid AE1 gene promoter by using run-on transcription, RNase protection, transient transfection, and transgenic mouse assays. AE1 mRNA was transcribed at a higher level and maintained at a higher steady-state level than either ankyrin or beta-spectrin in mouse fetal liver cells. When linked to a human gamma-globin gene, two different AE1 promoters directed erythroid-specific expression of gamma-globin mRNA in 18 of 18 lines of transgenic mice. However, variegated expression of gamma-globin was observed in 14 of 18 lines. While there was a significant correlation between transgene copy number and the amount of gamma-globin mRNA in all 18 lines, the transgene mRNAs initiated upstream of the start site of the endogenous AE1 mRNA. Addition of the insulator element from 5'HS4 of the chicken beta-globin cluster to the AE1/gamma-globin transgene allowed position-independent, copy-number-dependent expression at levels similar to the AE1 transcription rate in six of six lines of transgenic mice. The mRNA from the insulated AE1/gamma-globin transgene mapped to the start site of the endogenous AE1 mRNA, and gamma-globin protein was expressed in 100% of erythrocytes in all lines. We conclude that the chicken beta-globin 5'HS4 element is necessary for full function of the AE1 promoter and that position effect variegation is associated with RNA transcription from the upstream start sites.
Insulators are DNA sequence elements that can serve in some cases as barriers to protect a gene against the encroachment of adjacent inactive condensed chromatin. Some insulators also can act as blocking elements to protect against the activating influence of distal enhancers associated with other genes. Although most of the insulators identified so far derive from Drosophila, they also are found in vertebrates. An insulator at the 5' end of the chicken beta-globin locus marks a boundary between an open chromatin domain and a region of constitutively condensed chromatin. Detailed analysis of this element shows that it possesses both enhancer blocking activity and the ability to screen reporter genes against position effects. Enhancer blocking is associated with binding of the protein CTCF; sites that bind CTCF are found at other critical points in the genome. Protection against position effects involves other properties that appear to be associated with control of histone acetylation and methylation. Insulators thus are complex elements that can help to preserve the independent function of genes embedded in a genome in which they are surrounded by regulatory signals they must ignore.
Stably integrated transgenes flanked by the chicken beta-globin HS4 insulator are protected against chromosomal position effects and gradual extinction of expression during long-term propagation in culture. To investigate the mechanism of action of this insulator, we used bisulfite genomic sequencing to examine the methylation of individual CpG sites within insulated transgenes, and compared this with patterns of histone acetylation. Surprisingly, although the histones of the entire insulated transgene are highly acetylated, only a specific region in the promoter, containing binding sites for erythroid-specific transcription factors, is highly protected from DNA methylation. This critical region is methylated in noninsulated and inactive lines. MBD3 and Mi-2, subunits of the Mi-2/NuRD repressor complex, are bound in vivo to these silenced noninsulated transgenes. In contrast, insulated cell lines do not show any enrichment of Mi-2/NuRD proteins very late in culture. In addition to the high levels of histone acetylation observed across the entire insulated transgene, significant peaks of H3 acetylation are present over the HS4 insulator elements. Targeted histone acetylation by the chicken beta-globin insulator occurs independently of gene transcription and does not require the presence of a functional enhancer. We suggest that this acetylation is in turn responsible for the maintenance of a region of unmethylated DNA over the promoter. Whereas DNA methylation often leads to histone deacetylation, here acetylation appears to prevent methylation.
The 1.2-kb DNA sequence element (5'HS4) at the 5' end of the chicken beta-globin locus has the two defining properties of an insulator: it prevents an "external" enhancer from acting on a promoter when placed between them ("enhancer blocking") and acts as a barrier to chromosomal position effect (CPE) when it surrounds a stably integrated reporter. We previously reported that a single CTCF-binding site in 5'HS4 is necessary and sufficient for enhancer blocking. We show here that a 250-bp "core" element from within 5'HS4 is sufficient to confer protection against silencing of transgenes caused by CPE. Further dissection of the core reveals that 5'HS4 is a compound element in which it is possible to separate enhancer blocking and barrier activities. We demonstrate that full protection against CPE is conferred by mutant 5'HS4 sequences from which the CTCF-binding site has been deleted. In contrast, mutations of four other protein binding sites within 5'HS4 result in varying reductions in the ability to protect against CPE. We find that binding sites for CTCF are neither necessary nor sufficient for protection against CPE. Comparison of the properties of 5'HS4 with those of other CTCF-binding enhancer-blocking elements suggests that CPE protection is associated with maintenance of a high level of histone acetylation near the insulator, conferred by insulator binding-proteins other than CTCF.
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A binding site for the transcription factor CTCF is responsible for enhancer-blocking activity in a variety of vertebrate insulators, including the insulators at the 5' and 3' chromatin boundaries of the chicken beta-globin locus. To date, no functional domain boundaries have been defined at mammalian beta-globin loci, which are embedded within arrays of functional olfactory receptor genes. In an attempt to define boundary elements that could separate these gene clusters, CTCF-binding sites were searched for at the most distal DNase I-hypersensitive sites (HSs) of the mouse and human beta-globin loci. Conserved CTCF sites were found at 5'HS5 and 3'HS1 of both loci. All of these sites could bind to CTCF in vitro. The sites also functioned as insulators in enhancer-blocking assays at levels correlating with CTCF-binding affinity, although enhancer-blocking activity was weak with the mouse 5'HS5 site. These results show that with respect to enhancer-blocking elements, the architecture of the mouse and human beta-globin loci is similar to that found previously for the chicken beta-globin locus. Unlike the chicken locus, the mouse and human beta-globin loci do not have nearby transitions in chromatin structure but the data suggest that 3'HS1 and 5'HS5 may function as insulators that prevent inappropriate interactions between beta-globin regulatory elements and those of neighboring domains or subdomains, many of which possess strong enhancers.