Hat1: the emerging cellular roles of a type B histone acetyltransferase.

Hat1 is the sole known example of a type B histone acetyltransferase. While it has long been presumed that type B histone acetyltransferases participate in the acetylation of newly synthesized histones during the process of chromatin assembly, definitive evidence linking these enzymes to this process has been scarce. This review will discuss recent results that have begun to shed light on the roles of Hat1 and also address several outstanding questions relating to the cellular function of this enzyme.

The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells.

MS-275 is a histone deacetylase (HDAC) inhibitor that has been reported to mediate its cytotoxic effect through generation of reactive oxygen species (ROS) in proliferating hematopoietic cell lines. We examined efficacy of MS-275 in nonproliferating chronic lymphocytic leukemia (CLL) cells from patients. In these cells, MS-275 demonstrated an in vitro LC(50) that was one log lower than for normal mononuclear cells. Following MS-275 treatment, histones H3 and H4 showed increased acetylation and HDAC enzymatic activity was reduced. Caspase-8, -9, and -3 were activated, and caspase substrates PARP and BID were cleaved. Additionally, FLICE-inhibitory protein (FLIP) was downmodulated following MS-275 incubation. MS-275 treatment caused detectable ROS generation after 15 h of incubation, which was blocked by the caspase inhibitor Z-VAD-fmk. Overexpression of Bcl-2 protein protected against MS-275-induced apoptosis. These data demonstrate that MS-275 is a promising therapy for the treatment of CLL, but that in contrast to previous reports, ROS generation does not precede commitment to apoptosis. Similar to many other therapeutic targets, MS-275-mediated apoptosis is reduced by overexpression of Bcl-2, justifying strategies to combine HDAC inhibitors with Bcl-2 antagonists.

Depsipeptide (FR 901228) promotes histone acetylation, gene transcription, apoptosis and its activity is enhanced by DNA methyltransferase inhibitors in AML1/ETO-positive leukemic cells.

In t(8;21) acute myeloid leukemia (AML), the AML1/ETO fusion protein promotes leukemogenesis by recruiting histone deacetylase (HDAC) and silencing AML1target genes important for hematopoietic differentiation. We hypothesized that depsipeptide (FR901228), a novel HDAC inhibitor evaluated in ongoing clinical trials, restores gene transcription and cell differentiation in AML1/ETO-positive cells. A dose-dependent increase in H3 and H4 histone acetylation was noted in depsipeptide-treated AML1/ETO-positive Kasumi-1 cells and blasts from a patient with t(8;21) AML. Consistent with this biological effect, we also showed a dose-dependent increase in cytotoxicity, expression of IL-3, here used as read-out for silenced AML1-target genes, upregulation of CD11b with other morphologic changes suggestive of partial cell differentiation in Kasumi-1 cells. Some of these biologic effects were also attained in other myeloid leukemia cell lines, suggesting that depsipeptide has differentiation and cytotoxic activity in AML cells, regardless of the underlying genomic abnormality. Notably, the activity of depsipeptide was enhanced by 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor (DNMT). These two agents in combination resulted in enhanced histone acetylation, IL-3 expression, and cytotoxicity, suggesting HDAC and DNMT activities as a potential dual target in future therapeutic strategies for AML1/ETO and other molecular subgroups of AML.

Type B histone acetyltransferase Hat1p participates in telomeric silencing.

Hat1p and Hat2p are the two subunits of a type B histone acetyltransferase from Saccharomyces cerevisiae that acetylates free histone H4 on lysine 12 in vitro. However, the role for these gene products in chromatin function has been unclear, as deletions of the HAT1 and/or HAT2 gene displayed no obvious phenotype. We have now identified a role for Hat1p and Hat2p in telomeric silencing. Telomeric silencing is the transcriptional repression of telomere-proximal genes and is mediated by a special chromatin structure. While there was no change in the level of silencing on a telomeric gene when the HAT1 or HAT2 gene was deleted, a significant silencing defect was observed when hat1Delta or hat2Delta was combined with mutations of the histone H3 NH(2)-terminal tail. Specifically, when at least two lysine residues were changed to arginine in the histone H3 tail, a hat1Delta-dependent telomeric silencing defect was observed. The most dramatic effects were seen when one of the two changes was in lysine 14. In further analysis, we found that a single lysine out of the five in the histone H3 tail was sufficient to mediate silencing. However, K14 was the best at preserving silencing, followed by K23 and then K27; K9 and K18 alone were insufficient. Mutational analysis of the histone H4 tail indicated that the role of Hat1p in telomeric silencing was mediated solely through lysine 12. Thus, in contrast to other histone acetyltransferases, Hat1p activity was required for transcriptional repression rather than gene activation.

The major cytoplasmic histone acetyltransferase in yeast: links to chromatin replication and histone metabolism.

We have isolated the predominant cytoplasmic histone acetyltransferase activity from Saccharomyces cerevisiae. This enzyme acetylates the lysine at residue 12 of free histone H4 but does not modify histone H4 when packaged in chromatin. The activity contains two proteins, Hat1p and Hat2p. Hat1p is the catalytic subunit of the histone acetyltransferase and has an intrinsic substrate specificity that modifies lysine in the recognition sequence GXGKXG. The specificity of the enzyme in the yeast cytoplasm is restricted relative to recombinant Hat1p suggesting that it is negatively regulated in vivo. Hat2p, which is required for high affinity binding of the acetyltransferase to histone H4, is highly related to Rbap48, which is a subunit of the chromatin assembly factor, CAF-1, and copurifies with the human histone deacetylase HD1. We propose that the Hat2p/Rbap48 family serve as escorts of histone metabolism enzymes to facilitate their interaction with histone H4.

The yeast EGD2 gene encodes a homologue of the alpha NAC subunit of the human nascent-polypeptide-associated complex.

Two Saccharomyces cerevisiae proteins of 21 and 27 kDa co-purify with a novel enhancer of Gal4p DNA binding activity (Egdp) [Parthun et al., Mol. Cell. Biol. 12 (1992) 5683-5689]. Mutations in the EGD1 gene encoding the 21-kDa protein (Egd1p) have been shown to affect the kinetics and extent of the Gal4p-mediated, galactose-induced activation of the GAL genes. Egd1p is homologous to human BTF3b, recently identified as the beta subunit of the heterodimeric nascent-polypeptide-associated complex (NAC) involved in ensuring signal-sequence-specific protein sorting and translocation [Wiedmann et al., Nature 370 (1994) 434-440]. We have cloned and characterized EGD2 encoding the 27-kDa protein and found that Egd2p is strikingly similar to the alpha subunit of human NAC. Yeast, therefore, contains a complex composed of Egd1p and Egd2p very similar to the NAC complex described in human cells. Disruption of EGD2, alone or in combination with an EGD1 disruption, causes no obvious phenotypes. The lack of phenotype, the high levels of EGD1 and EGD2 expression, and the identification of multiple human genes encoding NAC subunits suggest that the yeast EGD genes may be members of multigene families with redundant function.

The EGD1 product, a yeast homolog of human BTF3, may be involved in GAL4 DNA binding.

A variety of techniques, including filter binding, footprinting, and gel retardation, can be used to assay the transcriptional activator GAL4 (Gal4p) through the initial steps of its purification from yeast cells. Following DNA affinity chromatography, Gal4p still bound DNA selectively when assayed by filter binding or footprinting. However, the affinity-purified protein was no longer capable of forming a stable complex with DNA, as assayed by gel retardation. Mixing the purified Gal4p with the flowthrough fraction from the DNA affinity column restored gel retardation complex formation. Gel retardation assays were used to monitor the purification of a heat-stable Gal4p-DNA complex stabilization activity from the affinity column flowthrough. The activity coeluted from the final purification step with polypeptides of 21 and 27 kDa. The yeast gene encoding the 21-kDa protein was cloned on the basis of its N-terminal amino acid sequence. The gene, named EGD1 (enhancer of GAL4 DNA binding), encodes a highly basic protein (21% lysine and arginine) with a predicted molecular mass of 16.5 kDa. The amino acid sequence of the EGD1 product, Egd1p, is highly similar to that of the human protein BTF3 (X. M. Zheng, D. Black, P. Chambon, and J. M. Egly, Nature [London] 344:556-559, 1990). Although an egd1 null mutant was viable and Gal+, induction of the galactose-regulated genes in the egd1 mutant strain was significantly reduced when cells were shifted from glucose to galactose.

A transcriptionally active form of GAL4 is phosphorylated and associated with GAL80.

The GAL4 activator and GAL80 repressor proteins regulate the expression of yeast genes in response to galactose. A complex of the two proteins isolated from glucose-grown cells is inactive in an in vitro transcription reaction but binds DNA and blocks activation by the GAL4-VP16 chimeric activator. The complex purified from galactose-grown cells contains a mixture of phosphorylated and unphosphorylated forms of GAL4. The galactose-induced form of GAL4 activates in vitro transcription to levels similar to those seen with GAL4-VP16. The induced GAL4 complex is indistinguishable in size and apparent shape from the uninduced complex, consistent with a continued association with GAL80. These results confirm in vivo analyses that correlate GAL4 phosphorylation with galactose induction and support a model of transcriptional activation that does not require GAL80 dissociation.

Purification and characterization of the yeast transcriptional activator GAL4.

We have purified extensively the transcriptional activator, GAL4, from a yeast strain overexpressing the gene product from the ADH1 promoter. Our purification followed GAL4 activity by its binding to a specific DNA target sequence, using filter binding assays. No specific binding activity was detected in extracts from a strain containing a disrupted copy of the GAL4 gene. The purification protocol included fractionation of a whole cell extract by ion-exchange and DNA-affinity chromatography on a column containing a 17-base pair oligomer encoding a near consensus GAL4 binding site. Two polypeptides co-eluted with the GAL4 DNA binding activity from the DNA-affinity column. One had an apparent molecular mass of 99 kDa (the predicted size of the GAL4 protein) and cross-reacted with antibodies raised against GAL4 epitopes from fusion proteins expressed in bacterial cells. The second polypeptide did not cross-react with the anti-GAL4 antibody and is presumed to be the GAL80 transcriptional repressor based on its size (48 kDa) and known physical association with the GAL4 protein. GAL4 binding activity elutes from a gel filtration column as a 155-kDa species suggesting that it exists in solution in a heterodimer complex of one GAL4 and one GAL80 molecule. The dissociation constant of the DNA-affinity-purified GAL4-GAL80 complex for a 900-base pair DNA fragment containing the UASGAL element from the GAL1-GAL10 divergent promoter was, Kd(effective) (0.15 M KCl) = 2.4 x 10(-9) M.