SUMMARY Structural changes in specific chromatin domains are essential to the orderly progression of numerous nuclear processes, including transcription. We report that the nuclear protein NSBP1 (HMGN5), a recently discovered member of the HMGN nucleosome-binding protein family, is specifically targeted by its C-terminal domain to nucleosomes in euchromatin. We find that the interaction of NSBP1 with nucleosomes alters the compaction of cellular chromatin and that in living cells, NSBP1 interacts with linker histones. We demonstrate that the negatively charged C-terminal domain of NSBP1 interacts with the positively charged C-terminal domain of H5 and that NSBP1 counteracts the linker histone-mediated compaction of a nucleosomal array. Dysregulation of the cellular levels of NSBP1 alters the transcription level of numerous genes. We suggest that mouse NSBP1 is an architectural protein that binds preferentially to euchromatin and modulates the fidelity of the cellular transcription profile by counteracting the chromatin-condensing activity of linker histones.

Expression of the ATP-binding cassette transporter ABCB6 has been associated with multiple cellular functions, including resistance to several cytotoxic agents, iron homeostasis, and porphyrin transport. To further elucidate its physiological function and/or role in drug resistance, we determined the subcellular location of ABCB6. Using three novel ABCB6-specific antibodies, Western blot analysis of cells expressing cDNA-derived or endogenous ABCB6 revealed two distinct molecular weight forms. Confocal microscopy indicates that the protein localizes to both mitochondria and the plasma membrane. Differential centrifugation revealed that the lower molecular weight form predominantly resides in the mitochondria, while the larger protein form is more abundant in the plasma membrane. Preliminary studies indicate that ABCB6 is functionally relevant in the plasma membrane, where its expression prevents the accumulation of specific porphyrins in the cell. Digitonin solubilization of mitochondria demonstrated that ABCB6 is present in the outer mitochondrial membrane, while back-titration assays with the ABCB6-specific antibodies reveal that the nucleotide binding domain of ABCB6 is cytoplasmic. These studies are the first to demonstrate that ABCB6 exists in two molecular weight forms, is localized to both the outer mitochondrial membrane and the plasma membrane, and plays a functional role in the plasma membrane.

Multidrug resistance mechanisms underlying the intractability of malignant melanomas remain largely unknown. In this study, we demonstrate that the development of multidrug resistance in melanomas involves subcellular sequestration of intracellular cytotoxic drugs such as cis-diaminedichloroplatinum II (cisplatin; CDDP). CDDP is initially sequestered in subcellular organelles such as melanosomes, which significantly reduces its nuclear localization when compared with nonmelanoma͞KB-3-1 epidermoid carcinoma cells. The melanosomal accumulation of CDDP remarkably modulates melanogenesis through a pronounced increase in tyrosinase activity. The altered melanogenesis manifested an Ϸ8-fold increase in both intracellular pigmentation and extracellular transport of melanosomes containing CDDP. Thus, our experiments provide evidence that melanosomes contribute to the refractory properties of melanoma cells by sequestering cytotoxic drugs and increasing melanosome-mediated drug export. Preventing melanosomal sequestration of cytotoxic drugs by inhibiting the functions of melanosomes may have great potential as an approach to improving the chemosensitivity of melanoma cells.cancer ͉ melanosomes ͉ skin ͉ tumor therapy ͉ multidrug resistance

Erythropoiesis is dependent on the activity of transcription factors, including the erythroid-specific erythroid Kruppel-like factor (EKLF). ChIP followed by massively parallel sequencing (ChIP-Seq) is a powerful, unbiased method to map transfactor occupancy. We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts. We correlated these results with the nuclear distribution of EKLF, RNA-Seq analysis of the transcriptome, and the occupancy of other erythroid transcription factors. In progenitor cells, EKLF is found predominantly at the periphery of the nucleus, where EKLF primarily occupies the promoter regions of genes and acts as a transcriptional activator. In erythroblasts, EKLF is distributed throughout the nucleus, and erythroblast-specific EKLF occupancy is predominantly in intragenic regions. In progenitor cells, EKLF modulates general cell growth and cell cycle regulatory pathways, whereas in erythroblasts EKLF is associated with repression of these pathways. The EKLF interactome shows very little overlap with the interactomes of GATA1, GATA2, or TAL1, leading to a model in which EKLF directs programs that are independent of those regulated by the GATA factors or TAL1. (Blood. 2011;118(17):e139-e148) IntroductionMore than 2 million red blood cells are released into the circulation every second by a multistep process known as erythropoiesis, 1 which is accompanied by significant changes in the RNA expression profile. 2 The erythroid-specific DNA binding protein erythroid Kruppel-like factor (Klf1; EKLF), the founding member of the mammalian Kruppel/Sp1-like family of C2H2-type zinc finger DNA binding proteins, plays a significant role in this process. 3,4 Klf1 mRNA and EKLF are expressed in both erythroid progenitor cells and terminally differentiating erythrocytes. 5,6 In cell lines and in vitro, EKLF has been demonstrated to physically and functionally interact with DNA at a conserved CCNCNCCCN motif and with additional protein cofactors. 7,8 In EKLF-deficient (Klf1 Ϫ/Ϫ ) mice, definitive fetal liver erythroid progenitor cells fail to progress to erythroblasts, 9 leading to anemia and lethality by embryonic day 15. 10,11 Genome-wide mRNA profiling of WT and Klf1 Ϫ/Ϫ erythroid cells has revealed significant dysregulation of Ͼ 3000 mRNAs. 9,12-15 Two-thirds of the dysregulated transcripts were present at reduced levels in Klf1 Ϫ/Ϫ cells, consistent with the role of EKLF as a transcriptional activator, whereas one-third were present at increased levels, consistent with the role of EKLF as a transcriptional repressor. 16 Chromatin immunoprecipitation (ChIP) demonstrated EKLF occupancy at selected sites in genes encoding erythrocyte proteins AHSP, ankyrin, ␤-spectrin, Band 3, and dematin, all of which are down-regulated in EKLF-deficient cells. 9,12-15 EKLF also participates in chromatin modification and DNase hypersensitive site formation through interactions with CBP/p300 and an SWI/SNFrelated chromatin remodeling complex. 17,18 For example,...

ING2 is a candidate tumor suppressor gene that can activate p53 by enhancing its acetylation. Here, we demonstrate that ING2 is also involved in p53-mediated replicative senescence. ING2 protein expression increased in late-passage human primary cells, and it colocalizes with serine 15-phosphorylated p53. ING2 and p53 also complexed with the histone acetyltransferase p300. ING2 enhanced the interaction between p53 and p300 and acted as a cofactor for p300-mediated p53 acetylation. The level of ING2 expression directly modulated the onset of replicative senescence. While overexpression of ING2 induced senescence in young fibroblasts in a p53-dependent manner, expression of ING2 small interfering RNA delayed the onset of senescence. Hence, ING2 can act as a cofactor of p300 for p53 acetylation and thereby plays a positive regulatory role during p53-mediated replicative senescence.

Determination of the expression and spatial distribution of molecular epitopes, or antigens, in patient tissue specimens has substantially improved the pathologist's ability to classify disease processes. Certain disease pathophysiologies are marked by characteristic increased or decreased expression of developmentally controlled antigens, defined as Cluster of Differentiation markers, that currently form the foundation for understanding lymphoid malignancies. While chromogens and organic fluorophores have been utilitized for some time in immunohistochemical analyses, developments in synthetic, inorganic fluorophore semiconductors, namely quantum dots, offer a versatile alternative reporter system. Quantum dots are stable fluorophores, are resistant to photobleaching, and are attributed with wide excitation ranges and narrow emission spectra. To date, routinely processed, formalin-fixed tissues have only been probed with two quantum dot reporters simultaneously. In the present study, streptavidin-conjugated quantum dots with distinct emission spectra were tested for their utility in identifying a variety of differentially expressed antigens (surface, cytoplasmic, and nuclear). Slides were analyzed using confocal laser scanning microscopy, which enabled with a single excitation wavelength (488 nm argon laser) the detection of up to seven signals (streptavidin-conjugated quantum dots 525, 565, 585, 605, 655, 705 and 805 nm) plus the detection of 4'6-DiAmidino-2-PhenylIndole with an infra-red laser tuned to 760 nm for two photon excitation. Each of these signals was specific for the intended morphologic immunohistochemical target. In addition, five of the seven streptavidin-conjugated quantum dots tested (not streptavidin-conjugated quantum dots 585 or 805 nm) were used on the same tissue section and could be analyzed simultaneously on routinely processed formalin-fixed, paraffin-embedded sections. Application of this multiplexing method will enable investigators to explore the clinically relevant multidimensional cellular interactions that underlie diseases, simultaneously. Keywords: immunofluorescence; quantum dots; immunohistochemistry; multiplexing; multispectral; confocal microscopy For some time, the application of antibodies in the immunohistochemical staining of tissues has markedly improved the classification and diagnosis of disease processes. Nowhere is this more evident than in the lymphoid malignancies, wherein the identification of differentiation antigens, also called CD markers, has fundamentally improved classification of diseases and elucidated the underlying pathophysiology. A constant infusion of new technologies into pathology practice has enabled these changes, including monoclonal antibodies, streptavidin/biotin interactions, antigen retrieval, organic fluorophores, and enzymatic amplification strategies. A novel nanotechnology, the quantum dot, an inorganic fluorophore, promises to offer the next technological breakthrough in the imaging of patient tissues. Quantum dots have recently been ci...

Aims/Hypothesis Translation of genetic association signals into molecular mechanisms for diabetes has been slow. The glucokinase regulatory protein (GKRP) P446L variant, associated with inverse modulation of glucose- and lipid-related traits, has been shown to alter the kinetics of glucokinase (GCK) inhibition. As GCK inhibition is associated with nuclear sequestration, we aimed to determine whether this variant also alters the direct interaction between GKRP and GCK and their intra-cellular localization. Methods Fluorescently-tagged rat and human wild-type or P446L-GKRP and GCK were transiently transfected into HeLa cells and mouse primary hepatocytes. Whole-cell and nuclear fluorescence was quantified in individual cells exposed to low (5.5mmol/l) or high (25mmol/l) glucose conditions. Interaction between GCK and GKRP were measured by sensitized emission-based FRET efficiency (FRETN). Results P446L-GKRP had a decreased degree of nuclear localization, ability to sequester GCK, and direct interaction with GCK as measured by FRET compared to WT-GKRP. Decreased interaction was observed between WT-GKRP and GCK at high (25mmol/l) compared to low glucose (5.5mmol/l) but not between P446L-GKRP and GCK. Rat WT-GKRP and P446L-GKRP behaved quite differently: both variants responded to high glucose by diminished sequestration of GCK but showed no effect of the P446L variant on nuclear localization or GCK sequestration. Conclusions Our study suggests the common human GKRP-P446L variant results in elevated hepatic glucose uptake and disposal by increasing active, cytosolic GCK. This would increase hepatic lipid biosynthesis but decrease fasting plasma glucose concentrations and provides a potential mechanism for the protective effect of this allele on type 2 diabetes risk.

The C1 domain zinc finger structure is highly conserved among the protein kinase C (PKC) superfamily members. As the interaction site for the second messenger sn-1,2-diacylglycerol (DAG) and for the phorbol esters, the C1 domain has been an important target for developing selective ligands for different PKC isoforms. However, the C1 domains of the atypical PKC members are DAG/phorbol ester-insensitive. Compared with the DAG/phorbol ester-sensitive C1 domains, the rim of the binding cleft of the atypical PKC C1 domains possesses four additional positively charged arginine residues (at positions 7, 10, 11, and 20). In this study, we showed that mutation to arginines of the four corresponding sites in the C1b domain of PKC␦ abolished its high potency for phorbol 12,13-dibutyrate in vitro, with only marginal remaining activity for phorbol 12-myristate 13-acetate in vivo. We also demonstrated both in vitro and in vivo that the loss of potency to ligands was cumulative with the introduction of the arginine residues along the rim of the binding cavity rather than the consequence of loss of a single, specific residue. Computer modeling reveals that these arginine residues reduce access of ligands to the binding cleft and change the electrostatic profile of the C1 domain surface, whereas the basic structure of the binding cleft is still maintained. Finally, mutation of the four arginine residues of the atypical PKC C1 domains to the corresponding residues in the ␦C1b domain conferred response to phorbol ester. We speculate that the arginine residues of the C1 domain of atypical PKCs may provide an opportunity for the design of ligands selective for the atypical PKCs.The protein kinase C (PKC) 3 isozymes comprise a family of serine/threonine protein kinases that are involved in the transduction of a large number of signals important for the regulation of proliferation, differentiation, apoptosis, and other physiological functions (1, 2). The family is divided into three subfamilies: 1) the conventional PKCs (cPKCs) ␣, ␤ I, ␤ II, and ␥; 2) the novel PKCs (nPKCs) ␦, ⑀, , and ; and 3) the atypical PKCs (aPKCs) and / (human PKC and mouse PKC are orthologs). These three subfamilies share a common requirement for phospholipids for their kinase activity but differ in their dependence on other activators (3). The cPKCs (␣, ␤ I, ␤ II, and ␥) meet the original definition of PKC as a Ca 2ϩ and phospholipid-dependent protein kinase. Indeed, they require phosphatidylserine (PS), Ca 2ϩ , and diacylglycerol (DAG) (or phorbol esters) for their activation. The nPKCs (␦, ⑀, , and ) require only DAG/phorbol ester and PS. The activation of aPKCs ( and /) is much different from that of the cPKCs and nPKCs. They require only PS but not Ca 2ϩ and DAG/phorbol ester (4). As an important second messenger, DAG plays the major role in transducing cellular signals to the PKC molecules (5). A highly conserved cysteine-rich motif (the so-called "C1 domain") in the regulatory region of the PKCs acts as the specific receptor for the DAG signal (6, 7). Al...