The replacement histone H2A.Z is variously reported as being linked to gene expression and preven... more The replacement histone H2A.Z is variously reported as being linked to gene expression and preventing the spread of heterochromatin in yeast, or concentrated at heterochromatin in mammals. To resolve this apparent dichotomy, affinity-purified antibodies against the N-terminal region of H2A.Z, in both a tri-acetylatedandnon-acetylatedstate,areused in native chromatin immmuno-precipitation experiments with mononucleosomes from three chicken cell types. The hyperacetylated species concentrates at the 50 end of active genes, both tissue specific and housekeeping but is absent from inactive genes, while the unacetylated form is absent from both active and inactive genes. A concentration of H2A.Z is also found at insulators under circumstances implying a link to barrier activity but not to enhancer blocking. Although acetylated H2A.Z is widespread throughout the interphase genome, at mitosis its acetylation is erased, the unmodified form remaining. Thus, although H2A.Z may operate as an e...
An antiserum raised against chemically acetylated histone H4 was found to recognize the epitope e... more An antiserum raised against chemically acetylated histone H4 was found to recognize the epitope epsilon-N-acetyl lysine. Affinity-purified antibodies were used to fractionate oligo- and mononucleosomal chromatin fragments from the nuclei of 15-day chicken embryo erythrocytes. Antibody-bound chromatin was found to contain elevated levels of acetylated core histones. On probing with sequences of alpha D globin, an actively transcribed gene, the antibody-bound chromatin was 15- to 30-fold enriched relative to the input chromatin. Using ovalbumin sequences as a probe, no enrichment was observed. The results demonstrate directly that transcriptionally active genes carry acetylated core histones.
The distribution of core histone acetylation across the chicken beta-globin locus has been mapped... more The distribution of core histone acetylation across the chicken beta-globin locus has been mapped in 15 day chicken embryo erythrocytes by immunoprecipitation of mononucleosomes with an antibody recognizing acetylated histones, followed by hybridization probing at several points in the locus. A continuum of acetylation was observed, covering both genes and intergenic regions. Using the same probes, the generalized sensitivity to DNase I was mapped by monitoring the disappearance of intact genomic restriction fragments from Southern transfers. Close correspondence between the 33 kb of sensitive chromatin and the extent of acetylation indicates that one role of the modification could be the generation and/or maintenance of the open conformation. The precision of acetylation mapping makes it a possible approach to the definition of chromosomal domain boundaries.
533 CORE HISTONE ACETYLATION MAPPING [29] room temperature and incubate in the same solution cont... more 533 CORE HISTONE ACETYLATION MAPPING [29] room temperature and incubate in the same solution containing 2 10 6 dpm of 125I-labeled protein A per milliliter for 2 hr. After three final washes with PBS-Tween buffer, dry blot and expose to X-ray film for autoradiography. ...
Chromatin immunoprecipitation (ChIP) is a technique widely used for determining the genomic locat... more Chromatin immunoprecipitation (ChIP) is a technique widely used for determining the genomic location of modified histones and other chromatin-associated factors. Here we describe the methodology we have used in our laboratory for the immunoprecipitation of chromatin isolated from cells in the absence of crosslinking. Chromatin released from nuclei by micrococcal nuclease digestion is centrifuged through sucrose gradients to allow selection of mono- or dinucleosomes. This allows a protein or modification at a particular gene or locus to be mapped at higher resolution than in a crosslinked ChIP experiment. Two methods for the immunoprecipitation of chromatin are described: a large-scale fractionation by which it is possible to visualize the proteins of the immunoprecipitate by polyacrylamide gel electrophoresis, PAGE and a small-scale method that is more appropriate when the quantity of chromatin is limited. The sequence content of DNA extracted from the immunoprecipitated chromatin is analyzed by hybridization of Southern or slot blots, or by quantitative polymerase chain reaction. Enrichment of particular sequences in the immunoprecipitated fraction reveals the presence and extent of the modification at this location.
The replacement histone H2A.Z is variously reported as being linked to gene expression and preven... more The replacement histone H2A.Z is variously reported as being linked to gene expression and preventing the spread of heterochromatin in yeast, or concentrated at heterochromatin in mammals. To resolve this apparent dichotomy, affinity-purified antibodies against the N-terminal region of H2A.Z, in both a tri-acetylatedandnon-acetylatedstate,areused in native chromatin immmuno-precipitation experiments with mononucleosomes from three chicken cell types. The hyperacetylated species concentrates at the 50 end of active genes, both tissue specific and housekeeping but is absent from inactive genes, while the unacetylated form is absent from both active and inactive genes. A concentration of H2A.Z is also found at insulators under circumstances implying a link to barrier activity but not to enhancer blocking. Although acetylated H2A.Z is widespread throughout the interphase genome, at mitosis its acetylation is erased, the unmodified form remaining. Thus, although H2A.Z may operate as an e...
An antiserum raised against chemically acetylated histone H4 was found to recognize the epitope e... more An antiserum raised against chemically acetylated histone H4 was found to recognize the epitope epsilon-N-acetyl lysine. Affinity-purified antibodies were used to fractionate oligo- and mononucleosomal chromatin fragments from the nuclei of 15-day chicken embryo erythrocytes. Antibody-bound chromatin was found to contain elevated levels of acetylated core histones. On probing with sequences of alpha D globin, an actively transcribed gene, the antibody-bound chromatin was 15- to 30-fold enriched relative to the input chromatin. Using ovalbumin sequences as a probe, no enrichment was observed. The results demonstrate directly that transcriptionally active genes carry acetylated core histones.
The distribution of core histone acetylation across the chicken beta-globin locus has been mapped... more The distribution of core histone acetylation across the chicken beta-globin locus has been mapped in 15 day chicken embryo erythrocytes by immunoprecipitation of mononucleosomes with an antibody recognizing acetylated histones, followed by hybridization probing at several points in the locus. A continuum of acetylation was observed, covering both genes and intergenic regions. Using the same probes, the generalized sensitivity to DNase I was mapped by monitoring the disappearance of intact genomic restriction fragments from Southern transfers. Close correspondence between the 33 kb of sensitive chromatin and the extent of acetylation indicates that one role of the modification could be the generation and/or maintenance of the open conformation. The precision of acetylation mapping makes it a possible approach to the definition of chromosomal domain boundaries.
533 CORE HISTONE ACETYLATION MAPPING [29] room temperature and incubate in the same solution cont... more 533 CORE HISTONE ACETYLATION MAPPING [29] room temperature and incubate in the same solution containing 2 10 6 dpm of 125I-labeled protein A per milliliter for 2 hr. After three final washes with PBS-Tween buffer, dry blot and expose to X-ray film for autoradiography. ...
Chromatin immunoprecipitation (ChIP) is a technique widely used for determining the genomic locat... more Chromatin immunoprecipitation (ChIP) is a technique widely used for determining the genomic location of modified histones and other chromatin-associated factors. Here we describe the methodology we have used in our laboratory for the immunoprecipitation of chromatin isolated from cells in the absence of crosslinking. Chromatin released from nuclei by micrococcal nuclease digestion is centrifuged through sucrose gradients to allow selection of mono- or dinucleosomes. This allows a protein or modification at a particular gene or locus to be mapped at higher resolution than in a crosslinked ChIP experiment. Two methods for the immunoprecipitation of chromatin are described: a large-scale fractionation by which it is possible to visualize the proteins of the immunoprecipitate by polyacrylamide gel electrophoresis, PAGE and a small-scale method that is more appropriate when the quantity of chromatin is limited. The sequence content of DNA extracted from the immunoprecipitated chromatin is analyzed by hybridization of Southern or slot blots, or by quantitative polymerase chain reaction. Enrichment of particular sequences in the immunoprecipitated fraction reveals the presence and extent of the modification at this location.
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