In eukaryotic cells, histone gene expression is one of the major events that mark entry into S phase. While this process is tightly linked to cell cycle position, how it is regulated by the cell cycle machinery is not known. Here we show that NPAT, a substrate of the cyclin E-Cdk2 complex, is associated with human replication-dependent histone gene clusters on both chromosomes 1 and 6 in S phase. We demonstrate that NPAT activates histone gene transcription and that this activation is dependent on the promoter elements (SSCSs) previously proposed to mediate cell cycle-dependent transcription. Cyclin E is also associated with the histone gene loci, and cyclin E-Cdk2 stimulates the NPAT-mediated activation of histone gene transcription. Thus, our results both show that NPAT is involved in a key S phase event and provide a link between the cell cycle machinery and activation of histone gene transcription.

To understand the mechanisms by which CDKs regulate cell cycle progression, it is necessary to identify and characterize the physiological substrates of these kinases. We have developed a screening method to identify novel CDK substrates. One of the cDNAs identified in the screen is identical to the recently isolated NPAT gene. Here we show that NPAT associates with cyclin E-CDK2 in vivo and can be phosphorylated by this CDK. The protein level of NPAT peaks at the G 1 /S boundary. Overexpression of NPAT accelerates S-phase entry, and this effect is enhanced by coexpression of cyclin E-CDK2. These results suggest that NPAT is a substrate of cyclin E-CDK2 and plays a role in S-phase entry.

IntroductionDiffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous disease comprising at least 3 major subtypes with distinct molecular, biologic, and clinical properties: activated B cell-like DLBCL (ABC-DLBCL), germinal center B cell-like DLBCL (GCB-DLBCL), and primary mediastinal B-cell lymphoma. 1,2 Although the overall cure rate for DLBCL reaches more than 50% with the current therapies such as R-CHOP (rituximab plus cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone/prednisolone), less than 40% of ABC-DLBCL patients are cured. [2][3][4] Therefore, new therapy approaches efficient for this and other DLBCL subtypes are highly desirable.The transcription factor NF-B controls the expression of a wide range of genes involved in cell proliferation, survival, stress response, angiogenesis, and inflammation. 5,6 NF-B activity is tightly regulated by multiple signaling pathways, and abnormal NF-B activation has been linked to cancer development and progression. [7][8][9] Constitutive NF-B activation has been observed in high frequency in all of the main DLBCL subtypes, especially in ABC-DLBCL, with more than 90% of the tumors showing nuclear NF-B, the hallmark of its activation. 9-14 A recent genomic study revealed that more than 60% of ABC-DLBCLs and approximately 30% of GCB-DLBCLs harbor somatic mutations in multiple components of NF-B signaling pathways, such as the BCR, CD40, and TLR pathways. 15 Significantly, it has been demonstrated that constitutive NF-B signaling is required for the proliferation and survival of ABC-DLBCL cell lines. 11,13,16,17 All of these observations suggest a primary role for constitutive NF-B signaling in the pathogenesis of DLBCL and, therefore, the NF-B signaling pathway may represent a rational therapeutic target in DLBCL. 9,11,18 Ubiquitination, the covalent attachment of the ubiquitin (Ub) molecule to target proteins, regulates diverse cellular processes. 19 Ubiquitination proceeds through a stepwise enzymatic cascade involving 3 classes of enzymes: a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The E1 enzyme activates Ub in an ATP-dependent manner and transfers the activated Ub to an E2 enzyme through the formation of a thioester bond between the carboxy terminus of Ub and the active site cysteine of the E2, generating an E2 and Ub thioester conjugate (referred to as E2ϳUb). The E2 then cooperates with an E3 to attach the Ub to a lysine residue of a substrate. Ub itself can serve as a substrate and the process can undergo multiple rounds, resulting in the formation of polyubiquitin chains. 19,20 Because Ub has 7 lysine residues and any one of them can be conjugated to another Ub, polyubiquitin chains of different linkages with distinct functional properties are formed in cells. For example, lysine 48 (K48)-linked polyubiquitin chains typically target substrates for proteasomal degradation, whereas K63-linked polyubiquitin chains function as scaffolds to assemble protein complexes in NF-B signaling and DNA repair. [...

Activation of the G(1) checkpoint following DNA damage leads to inhibition of cyclin E-Cdk2 and subsequent G(1) arrest in higher eucaryotes. Little, however, is known about the molecular events downstream of cyclin E-Cdk2 inhibition. Here we show that, in addition to the inhibition of DNA synthesis, ionizing radiation induces downregulation of histone mRNA levels in mammalian cells. This downregulation occurs at the level of transcription and requires functional p53 and p21(CIP1/WAF1) proteins. We demonstrate that DNA damage induced by ionizing radiation results in the suppression of phosphorylation of NPAT, an in vivo substrate of cyclin E-Cdk2 kinase and an essential regulator of histone gene transcription, and its dissociation from histone gene clusters in a p53/p21-dependent manner. Inhibition of Cdk2 activity by specific inhibitors in the absence of DNA damage similarly disperses NPAT from histone gene clusters and represses histone gene expression. Our results thus suggest that inhibition of Cdk2 activity following DNA damage results in the downregulation of histone gene transcription through dissociation of NPAT from histone gene clusters.

The Laguerre-Gaussian (LG) modes constitute a complete basis set for representing the transverse structure of a paraxial photon field in free space. Earlier workers have shown how to construct a device for sorting a photon according to its azimuthal LG mode index, which describes the orbital angular momentum (OAM) carried by the field. In this paper we propose and demonstrate a mode sorter based on the fractional Fourier transform to efficiently decompose the optical field according to its radial profile. We experimentally characterize the performance of our implementation by separating individual radial modes as well as superposition states. The reported scheme can, in principle, achieve unit efficiency and thus can be suitable for applications that involve quantum states of light. This approach can be readily combined with existing OAM mode sorters to provide a complete characterization of the transverse profile of the optical field. DOI: 10.1103/PhysRevLett.119.263602 In recent years, the transverse structure of optical photons has been established as a resource for storing and communicating quantum information [1]. In contrast to the twodimensional Hilbert space of polarization, it takes an unbounded Hilbert space to provide a mathematical representation for the transverse structure of the optical field. The large information capacity of structured photons has been recently utilized to enhance quantum key distribution [2][3][4][5] and a multitude of other applications [6][7][8][9][10]. The orbital angular momentum (OAM) modes have become increasingly popular for implementing multidimensional quantum states due to the relative ease in generation [11], manipulation [12], and characterization of these modes [13,14].Although the OAM modes provide a basis set for representing the azimuthal structure of photons, they cannot completely span the entire transverse state space, which encompasses an extra (radial) degree of freedom. The Laguerre-Gaussian (LG) mode functions provide a basis to fully represent the spatial structure of the transverse field [15][16][17]. These modes are characterized by two numbers, the radial mode index p ∈ f0; 1; 2; …g and the azimuthal mode index l ∈ f0; AE1; AE2; …g. While the azimuthal number l is well studied due to its association with the OAM of light [16]; the radial index p has so far remained relatively unexplored. The quantum coherence of photons in a superposition of orthogonal radial modes has been recently demonstrated in the context of quantum communication and high-dimensional entanglement [17][18][19]. The radial LG modes also hold a number of promising features, and have been studied in the contexts of self-healing [20], super-resolution [21], and hyperbolic momentum charge [22]. Despite the growing theoretical interest in utilizing the radial structure of photons, the experimental realizations have thus far been impeded because of the difficulty of measuring these modes.The initial step in characterization of the radial degree of freedom of light is to find a radial ...