Hideaki Tagami

Histone H3.1 and H3.3 Complexes Mediate Nucleosome Assembly Pathways Dependent or Independent of DNA Synthesis

Deposition of the major histone H3 (H3.1) is coupled to DNA synthesis during DNA replication and possibly DNA repair, whereas histone variant H3.3 serves as the replacement variant for the DNA-synthesis-independent deposition pathway. To address how histones H3.1 and H3.3 are deposited into chromatin through distinct pathways, we have purified deposition machineries for these histones. The H3.1 and H3.3 complexes contain distinct histone chaperones, CAF-1 and HIRA, that we show are necessary to mediate DNA-synthesis-dependent and -independent nucleosome assembly, respectively. Notably, these complexes possess one molecule each of H3.1/H3.3 and H4, suggesting that histones H3 and H4 exist as dimeric units that are important intermediates in nucleosome formation. This finding provides new insights into possible mechanisms for maintenance of epigenetic information after chromatin duplication.

CTCF Tethers an Insulator to Subnuclear Sites, Suggesting Shared Insulator Mechanisms across Species

Insulators can block an enhancer of one gene from activating a promoter on another nearby gene. Almost all described vertebrate insulators require binding of the regulatory protein CTCF for their activity. We show that CTCF copurifies with the nucleolar protein nucleophosmin and both are present at insulator sites in vivo. Furthermore, exogenous insulator sequences are tethered to the nucleolus in a CTCF-dependent manner. These interactions, quite different from those of the gypsy insulator element in Drosophila, may generate similar loop structures, suggesting a common theme and model for enhancer-blocking insulator action.

HJURP is a cell-cycle-dependent maintenance and deposition factor of CENP-A at centromeres.

The histone H3 variant CenH3, called CENP-A in humans, is central in centromeric chromatin to ensure proper chromosome segregation. In the absence of an underlying DNA sequence, it is still unclear how CENP-A deposition at centromeres is determined. Here, we purified non-nucleosomal CENP-A complexes to identify direct CENP-A partners involved in such a mechanism and identified HJURP. HJURP was not detected in H3.1- or H3.3-containing complexes, indicating its specificity for CENP-A. HJURP centromeric localization is cell cycle regulated, and its transient appearance at the centromere coincides precisely with the proposed time window for new CENP-A deposition. Furthermore, HJURP downregulation leads to a major reduction in CENP-A at centromeres and impairs deposition of newly synthesized CENP-A, causing mitotic defects. We conclude that HJURP is a key factor for CENP-A deposition and maintenance at centromeres.

Chk1 Is a Histone H3 Threonine 11 Kinase that Regulates DNA Damage-Induced Transcriptional Repression

DNA damage results in activation or suppression of transcription of a large number of genes. Transcriptional activation has been well characterized in the context of sequence-specific DNA-bound activators, whereas mechanisms of transcriptional suppression are largely unexplored. We show here that DNA damage rapidly reduces histone H3 Threonine 11 (T11) phosphorylation. This correlates with repression of genes, including cyclin B1 and cdk1. H3-T11 phosphorylation occurs throughout the cell cycle and is Chk1 dependent in vivo. Following DNA damage, Chk1 undergoes rapid chromatin dissociation, concomitant with reduced H3-T11 phosphorylation. Furthermore, we find that loss of H3-T11 phosphorylation correlates with reduced binding of the histone acetyltransferase GCN5 at cyclin B1 and cdk1 promoters and reduced H3-K9 acetylation. We propose a mechanism for Chk1 as a histone kinase, responsible for DNA-damage-induced transcriptional repression by loss of histone acetylation.

A CAF-1 dependent pool of HP1 during heterochromatin duplication

To investigate how the complex organization of heterochromatin is reproduced at each replication cycle, we examined the fate of HP1‐rich pericentric domains in mouse cells. We find that replication occurs mainly at the surface of these domains where both PCNA and chromatin assembly factor 1 (CAF‐1) are located. Pulse–chase experiments combined with high‐resolution analysis and 3D modeling show that within 90 min newly replicated DNA become internalized inside the domain. Remarkably, during this time period, a specific subset of HP1 molecules (α and γ) coinciding with CAF‐1 and replicative sites is resistant to RNase treatment. Furthermore, these replication‐associated HP1 molecules are detected in Suv39 knockout cells, which otherwise lack stable HP1 staining at pericentric heterochromatin. This replicative pool of HP1 molecules disappears completely following p150CAF‐1 siRNA treatment. We conclude that during replication, the interaction of HP1 with p150CAF‐1 is essential to promote delivery of HP1 molecules to heterochromatic sites, where they are subsequently retained by further interactions with methylated H3‐K9 and RNA.

The HP1α-CAF1-SetDB1-containing complex provides H3K9me1 for Suv39-mediated K9me3 in pericentric heterochromatin

Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1α (HP1α)–chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non‐nucleosomal histone H3. Therefore, the heterochromatic HP1α–CAF1–SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1α with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.

Differential acetylation of Tat coordinates its interaction with the co‐activators cyclin T1 and PCAF

The HIV‐1 transactivator protein, Tat, is an atypical transcriptional activator that functions through binding, not to DNA, but to a short leader RNA, TAR. Although details of its functional mechanism are still unknown, emerging findings suggest that Tat serves primarily to adapt co‐activator complexes such as p300, PCAF and P‐TEFb to the HIV‐1 long terminal repeat. Hence, an understanding of how Tat interacts with these cofactors is crucial. It has recently been shown that acetylation at a single lysine, residue 50, regulated the association of Tat with PCAF. Here, we report that in the absence of Tat acetylation, PCAF binds to amino acids 20–40 within Tat. Interestingly, acetylation of Tat at Lys28 abrogates Tat–PCAF interaction. Acetylation at Lys50 creates a new site for binding to PCAF and dictates the formation of a ternary complex of Tat–PCAF–P‐TEFb. Thus, differential lysine acetylation of Tat coordinates the interactions with its co‐activators, cyclin T1 and PCAF. Our results may help in understanding the ordered recruitment of Tat co‐activators to the HIV‐1 promoter.

A global repressor (Mlc) is involved in glucose induction of the ptsG gene encoding major glucose transporter in Escherichia coli

Glucose stimulates the expression of ptsG encoding the major glucose transporter in Escherichia coli. We isolated Tn10 insertion mutations that confer constitutive expression of ptsG. The mutated gene was identified as mlc, encoding a protein that is known to be a repressor for transcription of several genes involved in carbohydrate utilization. Expression of ptsG was eliminated in a mlc crp double‐negative mutant. The Mlc protein was overproduced and purified. In vitro transcription studies demonstrated that transcription of ptsG is stimulated by CRP–cAMP and repressed by Mlc. The action of Mlc is dominant over that of CRP–cAMP. DNase I footprinting experiments revealed that CRP–cAMP binds at two sites centred at −40.5 and −95.5 and that Mlc binds at two regions centred around −8 and −175. The binding of CRP–cAMP stimulated the binding of RNA polymerase to the promoter while Mlc inhibited the binding of RNA polymerase but not the binding of CRP–cAMP. Gel‐mobility shift assay indicated that glucose does not affect the Mlc binding to the ptsG promoter. Our results suggest that Mlc is responsible for the repression of ptsG transcription and that glucose modulates the Mlc activity by unknown mechanism.

Negative regulation of the pts operon by Mlc: mechanism underlying glucose induction in Escherichia coli

The pts operon of Escherichia coli consists of three genes ptsH, ptsI and crr, each encoding for central components of the phosphoenolpyruvate: carbohydrate phosphotransferase system, HPr, enzyme I and IIAGlc, respectively. Transcription of the pts operon is stimulated when glucose is present in the culture medium. One of the two major promoters, P0, is responsible for this glucose induction. However, no regulatory protein responsible for the glucose induction of the pts operon has been identified yet and molecular mechanism by which glucose stimulates the pts transcription is not known.

A human T-cell lymphotropic virus type 1 enhancer of Myc transforming potential stabilizes Myc-TIP60 transcriptional interactions.

ABSTRACT The human T-cell lymphotropic virus type 1 (HTLV-1) infects and transforms CD4+ lymphocytes and causes adult T-cell leukemia/lymphoma (ATLL), an aggressive lymphoproliferative disease that is often fatal. Here, we demonstrate that the HTLV-1 pX splice-variant p30II markedly enhances the transforming potential of Myc and transcriptionally activates the human cyclin D2 promoter, dependent upon its conserved Myc-responsive E-box enhancer elements, which are associated with increased S-phase entry and multinucleation. Enhancement of c-Myc transforming activity by HTLV-1 p30II is dependent upon the transcriptional coactivators, transforming transcriptional activator protein/p434 and TIP60, and it requires TIP60 histone acetyltransferase (HAT) activity and correlates with the stabilization of HTLV-1 p30II/Myc-TIP60 chromatin-remodeling complexes. The p30II oncoprotein colocalizes and coimmunoprecipitates with Myc-TIP60 complexes in cultured HTLV-1-infected ATLL patient lymphocytes. Amino acid residues 99 to 154 within HTLV-1 p30II interact with the TIP60 HAT, and p30II transcriptionally activates numerous cellular genes in a TIP60-dependent or TIP60-independent manner, as determined by microarray gene expression analyses. Importantly, these results suggest that p30II functions as a novel retroviral modulator of Myc-TIP60-transforming interactions that may contribute to adult T-cell leukemogenesis.