Alan W. Thorne

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.

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 Putative Cancer Stem Cell Marker USP22 Is a Subunit of the Human SAGA Complex Required for Activated Transcription and Cell-Cycle Progression

Polycomb genes encode critical regulators of both normal stem cells and cancer stem cells. A gene signature that includes Polycomb genes and additional genes coregulated with Polycomb genes was recently identified. The expression of this signature has been reported to identify tumors with the cancer stem cell phenotypes of aggressive growth, metastasis, and therapy resistance. Most members of this 11 gene signature encode proteins with well-defined roles in human cancer. However, the function of the signature member USP22 remains unknown. We report that USP22 is a previously uncharacterized subunit of the human SAGA transcriptional cofactor complex. Within SAGA, USP22 deubiquitylates histone H2B. Furthermore, USP22 is recruited to specific genes by activators such as the Myc oncoprotein, where it is required for transcription. In support of a functional role within the Polycomb/cancer stem cell signature, USP22 is required for appropriate progression through the cell cycle.

Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis

Myofibroblasts are critical cellular elements of wound healing generated at sites of injury by transdifferentiation of resident cells. A paradigm for this process is conversion of hepatic stellate cells (HSC) into hepatic myofibroblasts. Treatment of HSC with DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) blocked transdifferentiation. 5-azadC also prevented loss of IκBα and PPARγ expression that occurs during transdifferentiation to allow acquisition of proinflammatory and profibrogenic characteristics. ChIP analysis revealed IκBα promoter is associated with transcriptionally repressed chromatin that converts to an active state with 5-azadC treatment. The methyl-CpG-binding protein MeCP2 which promotes repressed chromatin structure is selectively detected in myofibroblasts of diseased liver. siRNA knockdown of MeCP2 elevated IκBα promoter activity, mRNA and protein expression in myofibroblasts. MeCP2 interacts with IκBα promoter via a methyl-CpG-dependent mechanism and recruitment into a CBF1 corepression complex. We conclude that MeCP2 and DNA methylation exert epigenetic control over hepatic wound healing and fibrogenesis

Pygmoid Australomelanesian Homo sapiens skeletal remains from Liang Bua, Flores: Population affinities and pathological abnormalities

Liang Bua 1 (LB1) exhibits marked craniofacial and postcranial asymmetries and other indicators of abnormal growth and development. Anomalies aside, 140 cranial features place LB1 within modern human ranges of variation, resembling Australomelanesian populations. Mandibular and dental features of LB1 and LB6/1 either show no substantial deviation from modern Homo sapiens or share features (receding chins and rotated premolars) with Rampasasa pygmies now living near Liang Bua Cave. We propose that LB1 is drawn from an earlier pygmy H. sapiens population but individually shows signs of a developmental abnormality, including microcephaly. Additional mandibular and postcranial remains from the site share small body size but not microcephaly.

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.

USP22, an hSAGA subunit and potential cancer stem cell marker, reverses the polycomb-catalyzed ubiquitylation of histone H2A

Initial studies of the mammalian hSAGA transcriptional coactivator complex identified the acetyltransferase hGCN5/PCAF as the only known enzymatic subunit. Recently we demonstrated that the ubiquitin hydrolase USP22 comprises a second enzymatic subunit of hSAGA, and that is required for activator-driven transcription. USP22 is expressed with polycomb ubiquitin ligases in an 11 gene signature that defines therapy-resistant tumors. At the biochemical level, these Polycomb proteins function as global transcriptional repressors by catalyzing the ubiquitylation of histone H2A. In yeast, the USP22 homolog functions as a transcriptional coactivator by removing ubiquitin from a distinct core histones, H2B. Given that USP22 is expressed in cancer as part of an 11 gene signature that includes transcriptional repressors which ubiquitylate H2A, it seemed possible that USP22 might activate transcription in part via the deubiquitylation of this same substrate. As reported here, biochemical analysis of the substrate specificity of USP22 reveals that it deubiquitylates histone H2A in addition to H2B. This finding supports a model in which the H2A ubiquitin hydrolase USP22 is coordinately expressed with Polycomb H2A ubiquitin ligases in order that the transcription of certain critical transforming genes be maintained in the face of the global repression mediated by Polycomb.

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.

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.