The replacement histone H2A.Z in a hyperacetylated form is a feature of active genes in the chicken

Abstract

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 triacetylated and non-acetylated state, are used in native chromatin immmuno-precipitation experiments with mononucleosomes from three chicken cell types. The hyperacetylated species concentrates at the 5' 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 epigenetic marker for active genes, its N-terminal acetylation does not.

Figure 1

(A and B) Characterization of anti-H2A.Z and anti-AcH2A.Z affinity purified antibodies. HB, butyrate-treated HeLa histones; CE, 15-day embryo chicken erythrocyte histones; rH2A.Z, recombinant H2A.Z. (C and D) Characterization of proteins from ChIPs using these affinity purified antibodies with nucleosomes from 10-day chicken brain tissue (10DCB). I, U, B: Input, Unbound, Bound histones; Ab, carry-over products of primary antibodies.

Figure 2

Distributions of H2A.Z and AcH2A.Z at two housekeeping genes, GAPDH and vimentin and the tissue-specific gene carbonic anhydrase in HD37 erythroblasts. The bottom panel shows four amplicons at inactive sequences in HD37 cells and 15DCE. B/I values (the ratio of the Bound to the Input signal for an amplicon) above the unity lines represent ‘fold enrichment’ achieved by the antibodies. I/B values are plotted below the unity lines when the Bound signal is less than that of the Input: this ratio represents ‘fold depletion’.

Figure 3

Distributions of H2A.Z, AcH2A.Z, AcH4, AcH3 and AcH2B at the lysozyme locus in HD37 erythroblasts. Vertical arrows = DNase I hypersensitive sites and vertical bars with numbers indicate amplicon positions. The two genes, their transcriptional status, the 5′ and 3′ matrix attachment regions (MARs) and the CpG island are indicated. B/I values (the ratio of the Bound to the Input signal for an amplicon) above the unity lines represent ‘fold enrichment’ achieved by the antibodies. I/B values are plotted below the unity lines when the Bound signal is less than that of the Input: this ratio represents ‘fold depletion’.

Figure 4

Distributions of H2A.Z, AcH2A.Z, AcH4, AcH3 and AcH2B at the β-globin locus/folate receptor gene in 15-day chicken embryo erythrocytes (15DCE), 10-day chicken brain tissue (10DCB) and HD37 chicken erythroblasts. Nomenclature as in Figure 3. B/I values (the ratio of the Bound to the Input signal for an amplicon) above the unity lines represent ‘fold enrichment’ achieved by the antibodies. I/B values are plotted below the unity lines when the Bound signal is less than that of the Input: this ratio represents ‘fold depletion’.

Figure 5

Mouse L929 cell nuclei stained with DAPI and three different antibodies: H2A.Z(N′) and AcH2A.Z raised to the N-terminal tail of H2A.Z (the present antibodies) and an antibody previously raised (8) to a C-terminal epitope, H2A.Z(C′).