C. David Allis

Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation

Abstract. We have generated and characterized a novel site-specific antibody highly specific for the phosphorylated form of the amino-terminus of histone H3 (Ser10). In this study, we used this antibody to examine in detail the relationship between H3 phosphorylation and mitotic chromosome condensation in mammalian cells. Our results extend previous biochemical studies by demonstrating that mitotic phosphorylation of H3 initiates nonrandomly in pericentromeric heterochromatin in late G2 interphase cells. Following initiation, H3 phosphorylation appears to spread throughout the condensing chromatin and is complete in most cell lines just prior to the formation of prophase chromosomes, in which a phosphorylated, but nonmitotic, chromosomal organization is observed. In general, there is a precise spatial and temporal correlation between H3 phosphorylation and initial stages of chromatin condensation. Dephosphorylation of H3 begins in anaphase and is complete immediately prior to detectable chromosome decondensation in telophase cells. We propose that the singular phosphorylation of the amino-terminus of histone H3 may be involved in facilitating two key functions during mitosis: (1) regulate protein-protein interactions to promote binding of trans-acting factors that “drive” chromatin condensation as cells enter M-phase and (2) coordinate chromatin decondensation associated with M-phase.

Steroid receptor coactivator-1 is a histone acetyltransferase.

Steroid receptors and coactivator proteins are thought to stimulate gene expression by facilitating the assembly of basal transcription factors into a stable preinitiation complex. What is not clear, however, is how these transcription factors gain access to transcriptionally repressed chromatin to modulate the transactivation of specific gene networks in vivo. The available evidence indicates that acetylation of chromatin in vivo is coupled to transcription and that specific histone acetyltransferases (HATs)target histones bound to DNA and overcome the inhibitory effect of chromatin on gene expression. The steroid-receptor coactivator SRC-1 is a coactivator for many members of the steroid-hormone receptor superfamily of ligand-inducible transcription factors. Here we show that SRC-1 possesses intrinsic histone acetyltransferase activity and that it also interacts with another HAT, p300/CBP-associated factor (PCAF). The HAT activity of SRC-1 maps to its carboxy-terminal region and is primarily specific for histones H3 and H4. Acetylation by SRC-1 and PCAF of histones bound at specific promoters may result from ligand binding to steroid receptors and could be a mechanism by which the activation functions of steroid receptors and associated coactivators enhance formation of a stable preinitiation complex, thereby increasing transcription of specific genes from transcriptionally repressed chromatin templates.

Roles of histone acetyltransferases and deacetylases in gene regulation

Acetylation of internal lysine residues of core histone N‐terminal domains has been found correlatively associated with transcriptional activation in eukaryotes for more than three decades. Recent discoveries showing that several transcriptional regulators possess intrinsic histone acetyltransferase (HAT) and deacetylase (HDAC) activities strongly suggest that histone acetylation and deacetylation each plays a causative role in regulating transcription. Intriguingly, several HATs have been shown an ability to acetylate nonhistone protein substrates (e.g., transcription factors) in vitro as well, suggesting the possibility that internal lysine acetylation of multiple proteins exists as a rapid and reversible regulatory mechanism much like protein phosphorylation. This article reviews recent developments in histone acetylation and transcriptional regulation. We also discuss several important, yet unanswered, questions. BioEssays 20:615–626, 1998.

The TAFII250 Subunit of TFIID Has Histone Acetyltransferase Activity

The transcription initiation factor TFIID is a multimeric protein complex composed of TATA box-binding protein (TBP) and many TBP-associated factors (TAF(II)s). TAF(II)s are important cofactors that mediate activated transcription by providing interaction sites for distinct activators. Here, we present evidence that human TAF(II)250 and its homologs in Drosophila and yeast have histone acetyltransferase (HAT) activity in vitro. HAT activity maps to the central, most conserved portion of dTAF(II)230 and yTAF(II)130. The HAT activity of dTAF(II)230 resembles that of yeast and human GCN5 in that it is specific for histones H3 and H4 in vitro. Our findings suggest that targeted histone acetylation at specific promoters by TAF(II)250 may be involved in mechanisms by which TFIID gains access to transcriptionally repressed chromatin.

Phosphorylation of Histone H3 Is Required for Proper Chromosome Condensation and Segregation

Phosphorylation of histone H3 at serine 10 occurs during mitosis in diverse eukaryotes and correlates closely with mitotic and meiotic chromosome condensation. To better understand the function of H3 phosphorylation in vivo, we created strains of Tetrahymena in which a mutant H3 gene (S10A) was the only gene encoding the major H3 protein. Although both micronuclei and macronuclei contain H3 in typical nucleosomal structures, defects in nuclear divisions were restricted to mitotically dividing micronuclei; macronuclei, which are amitotic, showed no defects. Strains lacking phosphorylated H3 showed abnormal chromosome segregation, resulting in extensive chromosome loss during mitosis. During meiosis, micronuclei underwent abnormal chromosome condensation and failed to faithfully transmit chromosomes. These results demonstrate that H3 serine 10 phosphorylation is causally linked to chromosome condensation and segregation in vivo and is required for proper chromosome dynamics.

Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation

Post-translational acetylation of the core histone amino-terminal tails has long been associated with both chromatin assembly and the regulation of gene expression. The recent identification and cloning of histone acetyltransferase genes represents a significant breakthrough in our understanding of how specific acetylation states are established. Ongoing characterization of these enzymes and their molecular cohorts supports a direct role for acetylation in a signaling pathway that modulates chromatin structure to create new patterns of transcription.

Overlapping but Distinct Patterns of Histone Acetylation by the Human Coactivators p300 and PCAF within Nucleosomal Substrates

A number of transcriptional coactivators possess intrinsic histone acetylase activity, providing a direct link between hyperacetylated chromatin and transcriptional activation. We have determined the core histone residues acetylated in vitro by recombinant p300 and PCAF within mononucleosomes. p300 specifically acetylates all sites of histones H2A and H2B known to be acetylated in bulk chromatin in vivo but preferentially acetylates lysines 14 and 18 of histone H3 and lysines 5 and 8 of histone H4. PCAF primarily acetylates lysine 14 of H3 but also less efficiently acetylates lysine 8 of H4. PCAF in its native form, which is present in a stable multimeric protein complex lacking p300/CBP, primarily acetylates H3 to a monoacetylated form, suggesting that PCAF-associated polypeptides do not alter the substrate specificity. These distinct patterns of acetylation by the p300 and PCAF may contribute to their differential roles in transcriptional regulation.

Histone acetylation and chromatin assembly: a single escort, multiple dances?

The remarkable flurry in identification of chromatin-modifying activities in recent times has been accompanied by unexpected connections between these activities and cellular transformation. The possible link between Rb function as a tumor suppressor and the function of p48 as a “histone escort” is intriguing, but is at present very speculative. However, disruption of interactions between a Gcn5p (HAT A) homolog, P/CAF, and its cofactors, p300 or CBP, by the product of the viral E1A oncogene is required for E1A-mediated cellular transformation (Yang et al. 1996xYang, X.J, Ogryzko, V.V, Nishikawa, J, Howard, B.H, and Nakatani, Y. Nature. 1996; 382: 319–324CrossRef | PubMed | Scopus (1184)See all ReferencesYang et al. 1996). Translocation of another putative acetylase, MOZ, and in-frame fusion to CBP is associated with specific subtypes of acute myeloid leukemias (Borrow et al. 1996xBorrow, J, Stanton, V.P Jr., Andresen, J.M, Becher, R, Behm, F.G, Chaganti, R.S.K, Civin, C.I, Disteche, C, Dube, I, Frischauf, A.M, Horsman, D, Mitelman, F, Volina, S, Watmore, A.E, and Housman, D.E. Nature Genet. 1996; 13: 33–41CrossRef | Scopus (518)See all ReferencesBorrow et al. 1996). MOZ is homologous to the yeast gene SAS2 (Something About Silencing) which is required for silencing in yeast (Reifsnyder et al. 1996xReifsnyder, C, Lowell, J, Clarke, A, and Pillus, L. Nature Genet. 1996; 13: 42–49CrossRef | Scopus (212)See all ReferencesReifsnyder et al. 1996), suggesting the human gene may also participate in silencing functions. Understanding the nature, regulation, and specificity of these activities is no longer just the pursuit of those interested in understanding the structure and function of chromatin, but is now directly relevant to our understanding of both normal cellular regulatory processes and abnormal processes which lead to oncogenesis.

Chromatin condensation: does histone H1 dephosphorylation play a role?

In this article we describe three distinct biological systems where histone H1 phosphorylation is uncoupled from mitosis and highly condensed chromatin is enriched in dephosphorylated forms of H1: the amitotic macronucleus of Tetrahymena, terminally differentiated avian erythrocytes and sea urchin sperm. Each system offers informative contrasts to the idea that H1 hyperphosphorylation is causally related to mitotic chromosome condensation. Assuming that higher order chromatin folding is primarily driven by electrostatic interactions between H1 and DNA, an alternative model is presented for the role of H1 phosphorylation in chromatin condensation.

Histone acetyltransferase activity and interaction with ADA2 are critical for GCN5 function in vivo

Yeast GCN5 is one component of a putative adaptor complex that includes ADA2 and ADA3 and functionally connects DNA‐bound transcriptional activators with general transcription factors. GCN5 possesses histone acetyltransferase (HAT) activity, conceptually linking transcriptional activation with enzymatic modification at chromatin. We have identified the minimal catalytic domain within GCN5 necessary to confer HAT activity and have shown that in vivo activity of GCN5 requires this domain. However, complementation of growth and transcriptional activation in gcn5− cells required not only the HAT domain of GCN5, but also interaction with ADA2. The bromodomain in GCN5 was dispensable for HAT activity and for transcriptional activation by strong activators; however, it was required for full complementation in other assays. Fusion of GCN5 to the bacterial lexA DNA binding domain activated transcription in vivo, and required both the HAT domain and the ADA2 interaction domain. These results suggest that both functions of GCN5, HAT activity and interaction with ADA2, are necessary for targeting and acetylation of nucleosomal histones.