Histone H4 lysine 20 monomethylation promotes transcriptional repression by L3MBTL1.

Lethal 3 malignant brain tumor 1 (L3MBTL1), a homolog of the Drosophila polycomb tumor suppressor l(3)mbt, contains three tandem MBT repeats (3xMBT) that are critical for transcriptional repression. We recently reported that the 3xMBT repeats interact with mono- and dimethylated lysines in the amino termini of histones H4 and H1b to promote methylation-dependent chromatin compaction. Using a series of histone peptides, we now show that the recognition of mono- and dimethylated lysines in histones H3, H4 and H1.4 (but not their trimethylated or unmodified counterparts) by 3xMBT occurs in the context of a basic environment, requiring a conserved aspartic acid (D355) in the second MBT repeat. Despite the broad range of in vitro binding, the chromatin association of L3MBTL1 mirrors the progressive accumulation of H4K20 monomethylation during the cell cycle. Furthermore, transcriptional repression by L3MBTL1 is enhanced by the H4K20 monomethyltransferase PR-SET7 (to which it binds) but not SUV420H1 (an H4K20 trimethylase) or G9a (an H3K9 dimethylase) and knockdown of PR-SET7 decreases H4K20me1 levels and the chromatin association of L3MBTL1. Our studies identify the importance of H4K20 monomethylation and of PR-SET7 for L3MBTL1 function.

Duration of nuclear NF-kappaB action regulated by reversible acetylation.

The nuclear expression and action of the nuclear factor kappa B (NF-kappaB) transcription factor requires signal-coupled phosphorylation and degradation of the IkappaB inhibitors, which normally bind and sequester this pleiotropically active factor in the cytoplasm. The subsequent molecular events that regulate the termination of nuclear NF-kappaB action remain poorly defined, although the activation of de novo IkappaBalpha gene expression by NF-kappaB likely plays a key role. Our studies now demonstrate that the RelA subunit of NF-kappaB is subject to inducible acetylation and that acetylated forms of RelA interact weakly, if at all, with IkappaBalpha. Acetylated RelA is subsequently deacetylated through a specific interaction with histone deacetylase 3 (HDAC3). This deacetylation reaction promotes effective binding to IkappaBalpha and leads in turn to IkappaBalpha-dependent nuclear export of the complex through a chromosomal region maintenance-1 (CRM-1)-dependent pathway. Deacetylation of RelA by HDAC3 thus acts as an intranuclear molecular switch that both controls the duration of the NF-kappaB transcriptional response and contributes to the replenishment of the depleted cytoplasmic pool of latent NF-kappaB-IkappaBalpha complexes.

Regulation of global acetylation in mitosis through loss of histone acetyltransferases and deacetylases from chromatin.

Histone acetylation, a reversible modification of the core histones, is widely accepted to be involved in remodeling chromatin organization for genetic reprogramming. Histone acetylation is a dynamic process that is regulated by two classes of enzymes, the histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although promoter-specific acetylation and deacetylation has received most of the recent attention, it is superimposed upon a broader acting and dynamic acetylation that profoundly affects many nuclear processes. In this study, we monitored this broader histone acetylation as cells enter and exit mitosis. In contrast to the hypothesis that HATs and HDACs remain bound to mitotic chromosomes to provide an epigenetic imprint for postmitotic reactivation of the genome, we observed that HATs and HDACs are spatially reorganized and displaced from condensing chromosomes as cells progress through mitosis. During mitosis, HATs and HDACs are unable to acetylate or deacetylate chromatin in situ despite remaining fully catalytically active when isolated from mitotic cells and assayed in vitro. Our results demonstrate that HATs and HDACs do not stably bind to the genome to function as an epigenetic mechanism of selective postmitotic gene activation. Our results, however, do support a role for spatial organization of these enzymes within the cell nucleus and their relationship to euchromatin and heterochromatin postmitotically in the reactivation of the genome.

The emerging role of class II histone deacetylases.

Histone acetylation and deacetylation play essential roles in modifying chromatin structure and regulating gene expression in all eukaryotes. Several histone acetyltransferases have been identified that act as transcriptional coactivators. In contrast, histone deacetylases (HDACs) are part of transcriptional corepressor complexes. Based on their similarity to known yeast factors, the human HDACs are grouped into three classes. Class I HDACs are similar to the yeast transcriptional repressor yRPD3, while class II HDACs are related to yHDA1 and class III HDACs to ySIR2. In this review, we focus on the biology of class II HDACs. These newly discovered enzymes have been implicated in cell differentiation and development, and many molecular details are emerging that shed light on class II HDAC function and regulation. We discuss the biological role of these factors in the context of physiological processes.

Human HDAC7 histone deacetylase activity is associated with HDAC3 in vivo.

Histone deacetylases (HDACs) are part of transcriptional corepressor complexes and play key roles in regulating chromatin structure. Three different classes of human HDACs have been defined based on their homology to HDACs found in Saccharomyces cerevisiae: RPD3 (class I), HDA1 (class II), and SIR2 (class III). Here we describe the identification and functional characterization of HDAC7, a new member of the human class II HDAC family. Although HDAC7 is localized mostly to the cell nucleus, it is also found in the cytoplasm, suggesting nucleocytoplasmic shuttling. The HDAC activity of HDAC7 maps to a carboxyl-terminal domain and is dependent on the interaction with the class I HDAC, HDAC3, in the cell nucleus. Cytoplasmic HDAC7 that is not bound to HDAC3 is enzymatically inactive. We provide evidence that the transcriptional corepressors SMRT and N-CoR could serve as critical mediators of HDAC7 activity by binding class II HDACs and HDAC3 by two distinct repressor domains. Different class II HDACs reside in the cell nucleus in stable and autonomous complexes with enzymatic activity, but the enzymatic activities associated with HDAC7 and HDAC4 rely on shared cofactors, including HDAC3 and SMRT/N-CoR.

Reduced mobility of the alternate splicing factor (ASF) through the nucleoplasm and steady state speckle compartments.

Compartmentalization of the nucleus is now recognized as an important level of regulation influencing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concentration to sites of active transcription, but splicing factors are also thought to exist in a freely diffusible state. In this study, we examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF-GFP) using time-lapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We find that ASF-GFP moves at rates up to 100 times slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of an RNA polymerase II transcription inhibitor and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites.

BCoR, a novel corepressor involved in BCL-6 repression.

BCL-6 encodes a POZ/zinc finger transcriptional repressor that is required for germinal center formation and may influence apoptosis. Aberrant expression of BCL-6 due to chromosomal translocations is implicated in certain subtypes of non-Hodgkin's lymphoma. The POZ domains of BCL-6 and several other POZ proteins interact with corepressors N-CoR and SMRT. Here we identify and characterize a novel corepressor BCoR (BCL-6 interacting corepressor), which is expressed ubiquitously in human tissues. BCoR can function as a corepressor when tethered to DNA and, when overexpressed, can potentiate BCL-6 repression. Specific class I and II histone deacetylases (HDACs) interact in vivo with BCoR, suggesting that BCoR may functionally link these two classes of HDACs. Strikingly, BCoR interacts selectively with the POZ domain of BCL-6 but not with eight other POZ proteins tested, including PLZF. Additionally, interactions between the BCL-6 POZ domain and SMRT, N-CoR, and BCoR are mutually exclusive. The specificity of the BCL-6/BCoR interaction suggests that BCoR may have a role in BCL-6-associated lymphomas.

A core SMRT corepressor complex containing HDAC3 and TBL1, a WD40-repeat protein linked to deafness.

The corepressor SMRT mediates repression by thyroid hormone receptor (TR) as well as other nuclear hormone receptors and transcription factors. Here we report the isolation of a novel SMRT-containing complex from HeLa cells. This complex contains transducin beta-like protein 1 (TBL1), whose gene is mutated in human sensorineural deafness. It also contains HDAC3, a histone deacetylase not previously thought to interact with SMRT. TBL1 displays structural and functional similarities to Tup1 and Groucho corepressors, sharing their ability to interact with histone H3. In vivo, TBL1 is bridged to HDAC3 through SMRT and can potentiate repression by TR. Intriguingly, loss-of-function TRbeta mutations cause deafness in mice and humans. These results define a new TR corepressor complex with a physical link to histone structure and a potential biological link to deafness.

Identification of a penta- and hexapeptide of islet amyloid polypeptide (IAPP) with amyloidogenic and cytotoxic properties.

Pancreatic amyloid is found in more than 95 % of type II diabetes patients. Pancreatic amyloid is formed by the aggregation of islet amyloid polypeptide (hIAPP or amylin), which is a 37-residue peptide. Because pancreatic amyloid is cytotoxic, it is believed that its formation is directly associated with the development of the disease. We recently showed that hIAPP amyloid formation follows the nucleation-dependent polymerization mechanism and proceeds via a conformational transition of soluble hIAPP into aggregated beta-sheets. Here, we report that the penta- and hexapeptide sequences, hIAPP(23-27) (FGAIL) and hIAPP(22-27) (NFGAIL) of hIAPP are sufficient for the formation of beta-sheet-containing amyloid fibrils. Although these two peptides differ by only one amino acid residue, they aggregate into completely different fibrillar assemblies. hIAPP(23-27) (FGAIL) fibrils self-assemble laterally into unusually broad ribbons, whereas hIAPP(22-27) (NFGAIL) fibrils coil around each other in a typical amyloid fibril morphology. hIAPP(20-27) (SNNFGAIL) also aggregates into beta-sheet-containing fibrils, whereas no amyloidogenicity is found for hIAPP(24-27) (GAIL), indicating that hIAPP(23-27) (FGAIL) is the shortest fibrillogenic sequence of hIAPP. Insoluble amyloid formation by the partial hIAPP sequences followed kinetics that were consistent with a nucleation-dependent polymerization mechanism. hIAPP(22-27) (NFGAIL), hIAPP(20-27) (SNNFGAIL), and also the known fibrillogenic sequence, hIAPP(20-29) (SNNFGAILSS) exhibited significantly lower kinetic and thermodynamic solubilities than the pentapeptide hIAPP(23-27) (FGAIL). Fibrils formed by all short peptide sequences and also by hIAPP(20-29) were cytotoxic towards the pancreatic cell line RIN5fm, whereas no cytotoxicity was observed for the soluble form of the peptides, a notion that is consistent with hIAPP cytotoxicity. Our results suggest that a penta- and hexapeptide sequence of an appropriate amino acid composition can be sufficient for beta-sheet and amyloid fibril formation and cytotoxicity and may assist in the rational design of inhibitors of pancreatic amyloid formation or other amyloidosis-related diseases.

Acetylation of the HIV-1 Tat protein by p300 is important for its transcriptional activity.

The human immunodeficiency virus 1 (HIV-1) Tat protein activates transcriptional elongation by recruiting the positive transcription elongation factor (pTEFb) complex to the TAR RNA element, which is located at the 5' extremity of all viral transcripts [1-3]. Tat also associates in vitro and in vivo with the transcriptional coactivator p300/CBP [4-6]. This association has been proposed to recruit the histone acetyltransferase (HAT) activity of p300 to the integrated HIV-1 promoter. We have observed that the purified p300 HAT domain acetylates recombinant Tat proteins in vitro and that Tat is acetylated in vivo. The major targets of acetylation by p300 are lysine residues (Lys50 and Lys51) in the arginine-rich motif (ARM) used by Tat to bind RNA and for nuclear import. Mutation of these residues in full-length recombinant Tat blocked its acetylation in vitro. Furthermore, mutation of these lysine residues to arginine markedly decreased the synergistic activation of he HIV promoter by Tat and p300 or by Tat and cyclin T1. These results demonstrate that acetylation of Tat by p300/CBP is important for its transcriptional activation of the HIV promoter.

A new family of human histone deacetylases related to Saccharomyces cerevisiae HDA1p.

Histone deacetylases are the catalytic subunits of multiprotein complexes that are targeted to specific promoters through their interaction with sequence-specific DNA-binding factors. We have cloned and characterized a new human cDNA, HDAC-A, with homology to the yeast HDA1 family of histone deacetylases. Analysis of the predicted amino acid sequence of HDAC-A revealed an open reading frame of 967 amino acids containing two domains: a NH2-terminal domain with no homology to known proteins and a COOH-terminal domain with homology to known histone deacetylases (42% similarity to RPD3, 60% similarity to HDA1). Three additional human cDNAs with high homology to HDAC-A were identified in sequence data bases, indicating that HDAC-A itself is a member of a new family of human histone deacetylases. The mRNA encoding HDAC-A was differentially expressed in a variety of human tissues. The expressed protein, HDAC-Ap, exhibited histone deacetylase activity and this activity mapped to the COOH-terminal region (amino acids 495-967) with homology to HDA1p. In immunoprecipitation experiments, HDAC-A interacted specifically with several cellular proteins, indicating that it might be part of a larger multiprotein complex.

Characterization of a human RPD3 ortholog, HDAC3.

Histone acetylation levels in cells result from a dynamic equilibrium between competing histone acetylases and deacetylases. Changes in histone acetylation levels occur during both transcriptional activation and silencing. Cloning of the cDNA for a human histone deacetylase (HDAC1) has shown that it represents a human ortholog of the yeast transcriptional regulator RPD3. We have screened the expressed sequence tag database (National Center for Biotechnology Information) with the yeast RPD3 sequence and identified a human ortholog of RPD3, HDAC3. This cDNA encodes a protein of 428 amino acids with 58% sequence identity with HDAC1p. By using a specific polyclonal antiserum recognizing the C-terminal domain of HDAC3p and Western blotting, we detected a single approximately 49-kDa band in several tumor cell lines. HDAC3p is expressed predominantly in the nuclear compartment. Immunoprecipitation experiments with either an antiserum against HDAC3p or an anti-FLAG antiserum and a flagged HDAC3 cDNA showed that HDAc3p exhibits deacetylase activity both on free histones and on purified nucleosomes. This deacetylase activity is inhibited by trichostatin, trapoxin, and butyrate in vitro to the same degree as the deacetylase activity associated to HDAC1p. These observations identify another member of a growing family of human HDAC genes.

Mutations in the tat gene are responsible for human immunodeficiency virus type 1 postintegration latency in the U1 cell line.

Previous reports have demonstrated that the U1 cell line, a model for postintegration latency, is defective at the level of Tat function and can be rescued by exogenously provided Tat protein. Sequence analysis of tat cDNAs from the U1 cell line identified two distinct forms of Tat, in agreement with the fact that this cell line contains two integrated human immunodeficiency (HIV) proviruses. One Tat cDNA lacked an ATG initiation codon, while the other contained an H-to-L mutation at amino acid 13 (H13-->L). Both tat cDNAs were defective in terms of transcriptional activation of long terminal repeat-luciferase reporter gene in transient-transfection experiments. Introduction of the H13-->L mutation in a wild-type tat background caused a severe reduction in transcriptional activation. Introduction of the same mutation in an infectious HIV molecular clone caused a severely defective phenotype which could be rescued when the HIV proviral DNA was transfected in a Jurkat cell line stably expressing the Tat protein (Jurkat-Tat) or in Jurkat cells treated with tumor necrosis factor alpha. Infectious virus stocks generated in Jurkat-Tat cells were used to infect Jurkat cells and exhibited severely impaired growth which could also be rescued by infecting Jurkat-Tat cells. These observations define tat mutations as a mechanism for HIV postintegration latency.

A point mutation in the HIV-1 Tat responsive element is associated with postintegration latency.

Study of the mechanism of HIV-1 postintegration latency in the ACH2 cell line demonstrates that these cells failed to increase HIV-1 production following treatment with exogenous Tat. Reasoning that the defect in ACH2 cells involves the Tat response, we analyzed the sequence of tat cDNA and Tat responsive element (TAR) from the virus integrated in ACH2. Tat cDNA sequence is closely related to that of HIV LAI, and the encoded protein is fully functional in terms of long terminal repeat (LTR) transactivation. Cloning of a region corresponding to the 5'-LTR from ACH2, however, identified a point mutation (C37 -> T) in TAR. This mutation impaired Tat responsiveness of the LTR in transient transfection assays, and the measured defect was complemented in cells that had been treated with tetradecanoyl phorbol acetate or tumor necrosis factor type alpha (TNF-alpha). A compensatory mutation in TAR (G28 -> A), designed to reestablish base pairing in the TAR hairpin, restored wild-type Tat responsiveness. When the (C37 -> T) mutation was introduced in an infectious clone of HIV-1, no viral production was measured in the absence of TNF-alpha, whereas full complementation was observed when the infection was conducted in the presence of TNF-alpha or when a compensatory mutation (G28 -> A) was introduced into TAR. These experiments identify a novel mutation associated with HIV-1 latency and suggest that alterations in the Tat-TAR axis can be a crucial determinant of the latent phenotype in infected individuals.