Colyn Crane-Robinson

A direct link between core histone acetylation and transcriptionally active chromatin

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.

Histone H3 lysine 4 methylation patterns in higher eukaryotic genes

Lysine residues within histones can be mono-, di - or tri-methylated. In Saccharomyces cerevisiae tri-methylation of Lys 4 of histone H3 (K4/H3) correlates with transcriptional activity, but little is known about this methylation state in higher eukaryotes. Here, we examine the K4/H3 methylation pattern at the promoter and transcribed region of metazoan genes. We analysed chicken genes that are developmentally regulated, constitutively active or inactive. We found that the pattern of K4/H3 methylation shows similarities to S. cerevisiae. Tri-methyl K4/H3 peaks in the 5′ transcribed region and active genes can be discriminated by high levels of tri-methyl K4/H3 compared with inactive genes. However, our results also identify clear differences compared to yeast, as significant levels of K4/H3 methylation are present on inactive genes within the β-globin locus, implicating this modification in maintaining a poised chromatin state. In addition, K4/H3 di-methylation is not genome-wide and di-methylation is not uniformly distributed throughout the transcribed region. These results indicate that in metazoa, di- and tri-methylation of K4/H3 is linked to active transcription and that significant differences exist in the genome-wide methylation pattern as compared with S. cerevisiae.

Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain.

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.

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

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.

Targeted and Extended Acetylation of Histones H4 and H3 at Active and Inactive Genes in Chicken Embryo Erythrocytes

Affinity-purified polyclonal antibodies recognizing the most highly acetylated forms of histones H3 and H4 were used in immunoprecipitation assays with chromatin fragments derived from 15-day chicken embryo erythrocytes by micrococcal nuclease digestion. The distribution of hyperacetylated H4 and H3 was mapped at the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the tissue-specific gene, carbonic anhydrase (CA). H3 and H4 acetylation was found targeted to the CpG island region at the 5′ end of both these genes, falling off in the downstream direction. In contrast, at the βA-globin gene, both H3 and H4 are highly acetylated throughout the gene and at the downstream enhancer, with a maximum at the promoter. Low level acetylation was observed at the 5′ end of the inactive ovalbumin gene. Run-on assays to measure ongoing transcription showed that theGAPDH and CA genes are transcribed at a much lower rate than the adult βA-globin gene. The extensive high level acetylation at the βA-globin gene correlates most simply with its high rate of transcription. The targeted acetylation of histones H3 and H4 at the GAPDH andCA genes is consistent with a role in transcriptional initiation and implies that transcriptional elongation does not necessarily require hyperacetylation.

Thyroid hormone‐regulated enhancer blocking: cooperation of CTCF and thyroid hormone receptor

The highly conserved, ubiquitously expressed, zinc finger protein CTCF is involved in enhancer blocking, a mechanism crucial for shielding genes from illegitimate enhancer effects. Interestingly, CTCF‐binding sites are often flanked by thyroid hormone response elements (TREs), as at the chicken lysozyme upstream silencer. Here we identify a similar composite site positioned upstream of the human c‐myc gene. For both elements, we demonstrate that thyroid hormone abrogates enhancer blocking. Relief of enhancer blocking occurs even though CTCF remains bound to the lysozyme chromatin. Furthermore, chromatin immunoprecipitation analysis of the lysozyme upstream region revealed that histone H4 is acetylated at the CTCF‐binding site. Loss of enhancer blocking by the addition of T3 led to increased histone acetylation, not only at the CTCF site, but also at the enhancer and the promoter. Thus, when TREs are adjacent to CTCF‐binding sites, thyroid hormone can regulate enhancer blocking, thereby providing a new property for what was previously thought to be constitutive enhancer shielding by CTCF.

DNA binding of a non-sequence-specific HMG-D protein is entropy driven with a substantial non-electrostatic contribution

The thermal properties of two forms of the Drosophila melanogaster HMG-D protein, with and without its highly basic 26 residue C-terminal tail (D100 and D74) and the thermodynamics of their non-sequence-specific interaction with linear DNA duplexes were studied using scanning and titration microcalorimetry, spectropolarimetry, fluorescence anisotropy and FRET techniques at different temperatures and salt concentrations. It was shown that the C-terminal tail of D100 is unfolded at all temperatures, whilst the state of the globular part depends on temperature in a rather complex way, being completely folded only at temperatures close to 0 degrees C and unfolding with significant heat absorption at temperatures below those of the gross denaturational changes. The association constant and thus Gibbs energy of binding for D100 is much greater than for D74 but the enthalpies of their association are similar and are large and positive, i.e. DNA binding is a completely entropy-driven process. The positive entropy of association is due to release of counterions and dehydration upon forming the protein/DNA complex. Ionic strength variation showed that electrostatic interactions play an important but not exclusive role in the DNA binding of the globular part of this non-sequence-specific protein, whilst binding of the positively charged C-terminal tail of D100 is almost completely electrostatic in origin. This interaction with the negative charges of the DNA phosphate groups significantly enhances the DNA bending. An important feature of the non-sequence-specific association of these HMG boxes with DNA is that the binding enthalpy is significantly more positive than for the sequence-specific association of the HMG box from Sox-5, despite the fact that these proteins bend the DNA duplex to a similar extent. This difference shows that the enthalpy of dehydration of apolar groups at the HMG-D/DNA interface is not fully compensated by the energy of van der Waals interactions between these groups, i.e. the packing density at the interface must be lower than for the sequence-specific Sox-5 HMG box.

The DNA sequence specificity of HMG boxes lies in the minor wing of the structure.

To establish the basis of sequence‐specific DNA recognition by HMG boxes we separately transferred the minor and major wings from the sequence‐specific HMG box of TCF1 alpha into their equivalent position in the non‐sequence‐specific box 2 of HMG1. Thus chimera THT1 contains the minor wing (of 11 N‐terminal and 25 C‐terminal residues) from the HMG box of TCF1 alpha and the major wing (the 45 residue central section) from HMG1 box 2, whilst the situation is reversed in chimera HTH1. The structural integrity of the two chimeric proteins was established by CD, NMR and their binding to four‐way junction DNA. Gel retardation and circular permutation assays showed that only chimera THT1, containing the TCF1 alpha minor wing, formed a sequence‐specific complex and bent the DNA. The bend angle was estimated to be 59 degrees for chimera THT1 and 52 degrees for the HMG box of TCF1 alpha. Our results, in combination with mutagenesis and other data, suggests a model for the DNA binding of HMG boxes in which the N‐terminal residues and part of helix 1 contact the minor groove on the outside of a bent DNA duplex.

CHROMATIN IMMUNOPRECIPITATION ASSAYS IN ACETYLATION MAPPING OF HIGHER EUKARYOTES

Publisher Summary This chapter describes the use of chromatin immunoprecipitation assays in the acetylation mapping of higher eukaryotes. Acetylation of specific lysine residues in the N-terminal domains of core histones is a biochemical marker of active genes. Affinity-purified polyclonal antibodies recognizing acetylated core histones (principally H4) and the epitope ɛ-acetyllysine have been used in chromatin immunoselection procedures (CHIP assays) with mononucleosomes and salt-soluble chromatin fragments generated by micrococcal nuclease to determine the spatial and temporal distribution of this reversible posttranslational modification. The methodologies described in the chapter use relatively large amounts of affinity-purified antibody and select large amounts of acetylated histone-rich chromatin. Alternative protocols using cross-linking, for example, with formaldehyde, have also been very successful. The overriding criterion for success in this approach is the quality of the antibody used—that is, its specificity, affinity, and purity.

A Short-range Gradient of Histone H3 Acetylation and Tup1p Redistribution at the Promoter of the Saccharomyces cerevisiae SUC2 Gene*

Chromatin immunoprecipitation assays are used to map H3 and H4 acetylation over the promoter nucleosomes and the coding region of the Saccharomyces cerevisiae SUC2 gene, under repressed and derepressed conditions, using wild type and mutant strains. In wild type cells, a high level of H3 acetylation at the distal end of the promoter drops sharply toward the proximal nucleosome that covers the TATA box, a gradient that become even steeper on derepression. In contrast, substantial H4 acetylation shows no such gradient and extends into the coding region. Overall levels of both H3 and H4 acetylation rise on derepression. Mutation of GCN5 or SNF2 lead to substantially reduced SUC2 expression; in gnc5 there is no reduction in basal H3 acetylation, but large reductions occur on derepression. SNF2 mutation has little effect on H3 acetylation, so SAGA and SWI/SNF recruitment seem to be independent events. H4 acetylation is little affected by either GCN5 or SNF2 mutation. In a double snf2/gcn5 mutant (very low SUC2 expression), H3 acetylation is at the minimal level, but H4 acetylation remains largely unaffected. Transcription is thus linked to H3 but not H4 acetylation. Chromatin immunoprecipitation assays show that Tup1p is evenly distributed over the four promoter nucleosomes in repressed wild type cells but redistributes upstream on derepression, a movement probably linked to its conversion from a repressor to an activator.