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Gary Felsenfeld

Publications and source records attributed to Gary Felsenfeld.

At least 19 recordsLinked to original sources
PubMed2004

Recruitment of histone modifications by USF proteins at a vertebrate barrier element.

The chicken beta-globin 5'HS4 insulator element acts as a barrier to the encroachment of chromosomal silencing. Endogenous 5'HS4 sequences are highly enriched with histone acetylation and H3K4 methylation regardless of neighboring gene expression. We report here that 5'HS4 elements recruit these histone modifications when protecting a reporter transgene from chromosomal silencing. Deletion studies identified a single protein binding site within 5'HS4, footprint IV, that is necessary for the recruitment of histone modifications and for barrier activity. We have determined that USF proteins bind to footprint IV. USF1 is present in complexes with histone modifying enzymes in cell extracts, and these enzymes specifically interact with the endogenous 5'HS4 element. Knockdown of USF1 expression leads to a loss of histone modification recruitment and subsequent encroachment of H3K9 methylation. We propose that barrier activity requires the constitutive recruitment of H3K4 methylation and histone acetylation at multiple residues to counteract the propagation of condensed chromatin structures.

Acetylation↗
PubMed2004

Transcription of Ig germline genes in single human B cells and the role of cytokines in isotype determination.

We have developed a critical test of the chromatin accessibility model of Ig isotype determination in which local unfolding of chromatin higher order structure (chromatin accessibility) in the region of specific germline genes in the H chain locus determines the Ab class to be expressed in the B cell. We show that multiple germline genes are constitutively transcribed in the majority of naive human B cells in a population. Thus, because chromatin in its higher order structure cannot be transcribed, the entire Ig H chain locus must be unfolded in naive B cells. We have also established that IL-4 and anti-CD40 act by enhancing transcription in the majority of cells, rather than by activating transcription in more of the cells. Transcriptional activity in the human H chain locus rules out the perturbation of chromatin higher order structure as a factor in isotype determination. We have also found that the levels of germline gene transcription cannot fully account for the levels of secretion of the different Ig isotypes, and that secretion of IgE, in particular, is suppressed relative to that of IgG.

B-Lymphocyte Subsets↗
PubMed2004

Antagonism between DNA hypermethylation and enhancer-blocking activity at the H19 DMD is uncovered by CpG mutations.

Imprinted expression at the H19-Igf2 locus depends on a differentially methylated domain (DMD) that acts both as a maternal-specific, methylation-sensitive insulator and as a paternal-specific site of hypermethylation. Four repeats in the DMD bind CCCTC-binding factor (CTCF) on the maternal allele and have been proposed to attract methylation on the paternal allele. We introduced point mutations into the DMD to deplete the repeats of CpGs while retaining CTCF-binding and enhancer-blocking activity. Maternal inheritance of the mutations left H19 expression and Igf2 imprinting intact, consistent with the idea that the DMD acts as an insulator. Conversely, paternal inheritance of these mutations disrupted maintenance of DMD methylation, resulting in biallelic H19 expression. Furthermore, an insulator was established on the paternally inherited mutated allele in vivo, reducing Igf2 expression and resulting in a 40% reduction in size of newborn offspring. Thus, the nine CpG mutations in the DMD showed that the two parental-specific roles of the H19 DMD, methylation maintenance and insulator assembly, are antagonistic.

Animals↗
PubMed2004

The 5'-HS4 chicken beta-globin insulator is a CTCF-dependent nuclear matrix-associated element.

The protein CTCF plays an essential role in the action of a widely distributed class of vertebrate enhancer-blocking insulators, of which the first example was found in a DNA sequence element, HS4, at the 5' end of the chicken beta-globin locus. HS4 contains a binding site for CTCF that is necessary and sufficient for insulator action. Purification of CTCF has revealed that it interacts with proteins involved in subnuclear architecture, notably nucleophosmin, a 38-kDa nucleolar phosphoprotein that is concentrated in nuclear matrix preparations. In this report we show that both CTCF and the HS4 insulator element are incorporated in the matrix; HS4 incorporation depends on the presence of an intact CTCF-binding site. However the DNA sequence in the neighborhood of HS4 is not like that of canonical matrix attachment regions, and its incorporation into the matrix fraction is not sensitive to ribonuclease, suggesting that the insulator is a distinct matrix-associated element.

Animals↗
PubMed2004

Both CTCF-dependent and -independent insulators are found between the mouse T cell receptor alpha and Dad1 genes.

The T cell rearrangement of the T cell receptor (TCR) genes TCRalpha and delta is specifically regulated by a complex interplay between enhancer elements and chromatin structure. The alpha enhancer is active in T cells and drives TCRalpha recombination in collaboration with a locus control region-like element located downstream of the Calpha gene on mouse chromosome 14. Twelve kb further down-stream lies another gene, Dad1, with a program of expression different from that of TCRalpha. The approximately 6-kb locus control region element lying between them contains multiple regulatory sites with a variety of roles in regulating the two genes. Previous evidence has indicated that among these there are widely distributed regions with enhancer blocking (insulating) activity. We have shown in this report that one of these sites, not previously examined, strongly binds the insulator protein CCTC-binding factor (CTCF) in vitro and in vivo and can function in an enhancer blocking assay. However, other regions within the 6-kb element that also can block enhancers clearly do not harbor CTCF sites and thus must reflect the presence of a previously undetected and distinct vertebrate insulator activity.

Acetylation↗
PubMed2004

Physical properties of a genomic condensed chromatin fragment.

We have studied the physical properties of a segment of condensed chromatin that lies upstream of the chicken beta-globin locus. This segment can be excised from an avian erythroleukemia cell line by restriction enzyme digestion and released from the nucleus as an essentially homogeneous fragment about 15.5 kbp long. Because of this homogeneity we could measure its sedimentation coefficient quite accurately by a combination of sucrose gradient and analytical ultracentrifugation. By measuring additionally the buoyant density of the cross-linked particle in CsCl we could deduce the total mass of the particle, hence its frictional coefficient, f, directly related to its shape. The measured value of f is consistent with a rod-like particle of the approximate length and diameter proposed earlier for the 30 nm chromatin fiber. The method is generally applicable to homogeneous particles of unique sequence at genomic abundance.

Animals↗
PubMed2004

CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species.

Insulators can block an enhancer of one gene from activating a promoter on another nearby gene. Almost all described vertebrate insulators require binding of the regulatory protein CTCF for their activity. We show that CTCF copurifies with the nucleolar protein nucleophosmin and both are present at insulator sites in vivo. Furthermore, exogenous insulator sequences are tethered to the nucleolus in a CTCF-dependent manner. These interactions, quite different from those of the gypsy insulator element in Drosophila, may generate similar loop structures, suggesting a common theme and model for enhancer-blocking insulator action.

Animals↗
PubMed2004

Chromatin remodeling by RNA polymerases.

Chromatin packages DNA tightly into the eukaryotic nucleus and maintains its proper functioning. Recent studies suggest the existence of two distinct mechanisms of progression of RNA polymerases through chromatin. The first is characteristic of eukaryotic RNA polymerase III, bacteriophage RNA polymerases, and probably ATP-dependent chromatin remodeling complexes. In this mechanism, nucleosomes are translocated without release of the octamer into solution. By contrast, transcription by RNA polymerase II (Pol II) involves displacement of one H2A-H2B dimer. Nucleosomes can present a barrier for transcribing Pol II that can be regulated in vivo. Analysis of the mechanisms of transcription through chromatin should provide important information about mechanisms of chromatin remodeling and gene regulation at the level of transcript elongation.

Adenosine Triphosphate↗
PubMed2003

Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9.

Transgenes stably integrated into cells or animals in many cases are silenced rapidly, probably under the influence of surrounding endogenous condensed chromatin. This gene silencing correlates with repressed chromatin structure marked by histone hypoacetylation, loss of methylation at H3 lysine 4, increase of histone H3 lysine 9 methylation as well as CpG DNA methylation at the promoter. However, the order and the timing of these modifications and their impact on transcription inactivation are less well understood. To determine the temporal order of these events, we examined a model system consisting of a transgenic cassette stably integrated in chicken erythroid cells. We found that histone H3 and H4 hypoacetylation and loss of methylation at H3 lysine 4 all occurred during the same window of time as transgene inactivation in both multicopy and low-copy-number lines. These results indicate that these histone modifications were the primary events in gene silencing. We show that the kinetics of silencing exclude histone H3 K9 and promoter DNA methylation as the primary causative events in our transgene system.

Acetylation↗
PubMed2003

Controlling the double helix.

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.

Animals↗
PubMed2003

Quantitative approaches to problems of eukaryotic gene expression.

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.

Animals↗
PubMed2003

Variegated expression from the murine band 3 (AE1) promoter in transgenic mice is associated with mRNA transcript initiation at upstream start sites and can be suppressed by the addition of the chicken beta-globin 5' HS4 insulator element.

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.

Animals↗
PubMed2003

Human beta-globin locus control region HS5 contains CTCF- and developmental stage-dependent enhancer-blocking activity in erythroid cells.

The human beta-globin locus contains five developmentally regulated beta-type globin genes. All five genes depend on the locus control region (LCR), located at the 5' end of the locus, for abundant globin gene transcription. The LCR is composed of five DNase I-hypersensitive sites (HSs), at least a subset of which appear to cooperate to form a holocomplex in activating genes within the locus. We previously tested the requirement for proper LCR polarity by inverting it in human beta-globin yeast artificial chromosome transgenic mice and observed reduced expression of all the beta-type globin genes regardless of developmental stage. This phenotype clearly demonstrated an orientation-dependent activity of the LCR, although the mechanistic basis for the observed activity was obscure. Here, we describe genetic evidence demonstrating that human HS5 includes enhancer-blocking (insulator) activity that is both CTCF and developmental stage dependent. Curiously, we also observed an attenuating activity in HS5 that was specific to the epsilon-globin gene at the primitive stage and was independent of the HS5 CTCF binding site. These observations demonstrate that the phenotype observed in the LCR-inverted locus was in part attributable to placing the HS5 insulator between the LCR HS enhancers (HS1 to HS4) and the promoter of the beta-globin gene.

Animals↗
PubMed2002

The insulation of genes from external enhancers and silencing chromatin.

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.

Animals↗
PubMed2002

The barrier function of an insulator couples high histone acetylation levels with specific protection of promoter DNA from methylation.

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.

Acetylation↗
PubMed2002

Position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities.

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.

Animals↗