LYSP100-Associated Nuclear Domains (LANDs - Blood

LYSP100-Associated Nuclear Domains (LANDs): Description of a New Class of Subnuclear Structures and Their Relationship to PML Nuclear Bodies

By Alexander L. Dent, Jonathan Yewdell, Francine Puvion-Dutilleul, Marcel H.M. Koken, Hugues de The, and Louis M. Staudt

The PML gene is fused to the retinoic acid receptor a (RARa) gene in t(15;17) acute promyelocytic leukemia (APL), creat- ing a PML-RARa fusion oncoprotein. The PML gene product has been localized to subnuclear dot-like structures vari- ously termed PODS, NDlOs, Kr bodies, or PML nuclear bodies (PML NBs). The present study describes the cloning of a lymphoid-restricted gene, LYSPIOO, that is homologous to another protein that localizes to PML NBs, SPIOO. In addition t o SPIOO homology regions, one LYSPIOO cDNA isoform contains a bromodomain and a PHD/lTC domain, which are present in a variety of transcriptional regulatory proteins. By immunofluorescence, LYSPlOO was localized to nuclear dots that were surprisingly largely nonoverlapping with PML NBs. However, a minority of LYSPIOO nuclear dots exactly colocalized with PML and SPIOO. We term the LYSPIOO

HE INTERPHASE CELL nucleus is compartmentalized into different structural domains that are important for

nuclear function (see Spector' for review). The nucleolus, the most prominent subnuclear domain, assembles ribosomes. Other classes of subnuclear domains, such as interchromatin granules, interchromatin fibrils, coiled bodies, and nuclear bodies (NBs), were initially defined as morphologically distinct structures by electron microscopy (EM). Subsequently, antibody probes have been used to distinguish subnuclear domains biochemically. For example, a variety of splicing factors have been localized to both interchromatin granules and coiled bodies. The processes of transcription, splicing, and DNA replication have been shown to take place in localized regions of the nucleus, although the relation of these events to the morphologically defined subnuclear structures remains to be fully elucidated.

An ultrastructurally distinct subnuclear structure, vari- ously termed the PML POD: ND10,5 or Kr body,6 has recently been studied intensively due to the observation that the PML protein localizes to this d ~ m a i n . ~ ~ . ~ Interest in the PML gene derives from its involvement in the characteristic t( 15; 17) translocation of acute promyelocytic leukemia (APL).*"' By immunofluorescence, the wild-type PML protein localizes to five to 15 dot-like or ring-like domains in

Using immuno-EM, PML was detected in annular or dot-like, electron-dense structures in the nucleoplasm, 0.3 to l pm in PML preferentially localized over the electron-dense outer ring of these structures. This PML-containing structure is morphologically similar to a subclass of NBs previously defined by EM (re- viewed in Brasch and Ochs"), and thus we will term this structure a PML NB in the present study. Although the normal function of PML NBs has remained elusive, a clue to their importance comes from the observation that DNA vi- ruses encode proteins that localize to PML NBs transiently and subsequently disrupt the structures.","

The t(l.5; 17) translocation of APL results in the expression of a fusion oncoprotein in which PML is fused to the retinoic acid receptor (Y (RA"Y).*"'~'"'' In APL cells, the characteristic PML NB structures are absent and, instead,

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the nucleus,2-4.7.1~.~~-15

Blood, Vol 88, No 4 (August 15), 1996: pp 1423-1436

structures "LANDs," for LYSP100-associated nuclear domains. Although LYSPIOO is expressed only in lymphoid cells, LANDs could be visualized in HeLa cells by transfection of a LYSPIOO cDNA. lmmunoelectron microscopy revealed LANDs t o be globular, electron-dense structures morphologically distinct from the annular structures characteristic of PML NBs. LANDs were most often found in the nucleoplasm, but were also found at the nuclear membrane and in the cytoplasm, suggesting that these structures may traffic between the cytoplasm and the nucleus. By double-immunogold labeling of PML and LYSPIOO, some LANDs were shown t o contain both PML and LYSPIOO. Thus, PML is localized t o a second subnuclear domain that is morphologically and biochemically distinct from PML NBs. 0 1996 by The American Society of Hematology.

PML is localized to a large number of much smaller nuclear APL patients treated with retinoic acid undergo a

clinical remission resulting from the differentiation of leuke- mic cells into mature granulocytes and a concomitant de- crease in proliferation (see Grignani et al'' for review). Inter- estingly, treatment of APL cells with retinoic acid causes reformation of the normal PML NB structures, leading to speculation that disruption of these subnuclear domains is integral to malignant transformation by PML RARcx.~.~.~ Wild-type PML can suppress the growth and malignant phe- notype of transformed cells,23-2s again suggesting that interference with PML function might contribute to the pathogen- esis of APL.

In the present study, we characterized a novel lymphoid- restricted protein, LYSPlOO, that is homologous to SPIOO, a protein that colocalizes with PML in PML NBs.~ SPlOO was originally defined as an antigen detected by autoantisera from patients with primary biliary and is one of several proteins of unknown function that are found in PML N B s . ~ . ~ ~ Using confocal microscopy and EM, we unex- pectedly found that LYSPlOO localized to a new class of subnuclear structures, which we term LANDs (LYSPIOO- associated nuclear domains), that are morphologically and spatially distinct from PML NBs. Interestingly, PML colo-

From the Metabolism Branch, National Cancer Institute, and the Laboratory of Viral Diseases, National Institute of Allergy and lnfec- tious Diseases, National Institutes of Health, Bethesda, MD; CNRS UPR 272, IRSC, Villejuif; and CNRS UPR 43 and the Department of Biochemistry, Hopital St Louis, Paris, France.

Submitted January 5, 1996; accepted April 8, 1996. Address reprint requests to Louis M. Staudt, MD, PhD, National

Institutes of Health, Bldg IO, Room 4Nl14. 9000 Rockville Pike, Bethesda, MD 20892.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section I734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/8804-03$3.00/0

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1424 DENT ET AL

calizes with LYSPlOO in s o m e LANDs, thus defining LANDs as a new PML-containing subnuclear structure.

MATERIALS AND METHODS

Cloning of LYSPIOO. Polymerase chain reaction (PCR)-ampli- fiable cDNA enriched for lymphoid-restricted genes was obtained by subtractive hybridization of cDNA from the Burkitt’s lymphoma line, Raji, with mRNA from the erythroleukemia line, K562.”’-3’ This amplified cDNA was used to construct a subtracted cDNA library as previously described.” Twelve hundred ninety clones from the resultant subtracted library were sequenced by automated DNA sequencing and evaluated for homology using the Basic Local Align- ment Search Tool (BLAST) algorithm. A partial cDNA clone, AD25 1 I , was found to have high homology with the amino-terminal region of SPIOO. The AD2511 partial LYSPIOO cDNA clone was used to screen a large-insert Lambda-ZAP (Stratagene La Jolla, CA) cDNA library prepared from the Raji cell line. cDNA library screen- ing and other DNA manipulations were performed using standard techniques.” DNA sequencing was performed using the Applied Biosystems (ABI; Foster City, CA) 373 Sequencer and PRISM (ABI) sequencing mixes. The sequences of LYSPIOO cDNA isoforms, LYSP100-A and LYSP100-B (Fig 2A), have been submitted to Gen- bank and assigned accession numbers U36499 and U36500, respectively.

Cloning ofalternute SPlOO isoforms. A PstI-XbaI fragment derived from LYSP100-A, which has sequences identical to the LYSP100-Blhuman nuclear phosphoprotein (HNPP)-like domain, was used to screen a HeLa cDNA library (Stratagene). A 2.7-kb cDNA clone obtained from this screen, termed HFPX, was sequenced and found to contain a short open reading frame (49 amino acids) with very high homology to the carboxy-terminus of LYSP100-B. The region of HFPX with the highest homology to LYSP100-B was used to design the following nested antisense PCR primers: (1 j outer primer, S’-ATATCCGAATTCCCTTCACCG- CACCACAGGTCAC-3’, and (2) inner primer, S‘-TGGAAGTAA- AGGAGCCTGAAAATC-3’. These primers were used for reverse transcriptase-PCR (cDNA cycle kit; Invitrogen, San Diego, CA) on Raji mRNA in combination with the following primers from the 3’ end of SPIOO: (1) outer primer, 5’-TGGAACGAATTCAGACCT- GAGTAATGGAGAAGAG-3’, and (2) inner primer. S’-CTTCAG- GAAACCTGCAGCTCATCC-3’. Specificity of the initial PCR product using the outer primers was verified using the inner primers. A 400-bp product from the initial amplification was digested with EcoRI and cloned into pBluescript (Stratagene). The sequence of the PCR product showed a novel alternatively spliced form of SPI 00, and the subcloned PCR product was then used as a probe to screen the Raji cDNA library described earlier. Four independent SPlOO cDNA clones derived from this screen were analyzed, and all four clones had similar open reading frames that were truncated with respect to the carboxy-terminus of LYSP100-B (Fig 2A). The DNA sequence of the SP100-B isoform has been submitted to Genbank and assigned accession number U36501.

Norfhern blot analysis. Human cell line and human tonsil RNAs were prepared and enriched for poly-(A)-containing mRNA selected using standard methods. Other human tissue mRNAs were purchased from Clontech (Palo Alto, CA). Human tonsils were obtained from Holy Cross Hospital (Silver Spring, MD) after elective tonsilecto- mies. Poly-(A)+ RNA (2 pg) was electrophoresed on a 1.5% agarose gel containing formaldehyde, and blotted onto Hybond-N (Amer- sham, Arlington Heights, IL). DNA probes were labeled with 32P- dCTP using a random primed labeling kit (Boehringer Mannheim, Indianapolis, IN). The LYSPlOO probe was a full-length EcoRI-Xhol LYSP100-A cDNA fragment. The human glyceraldehyde phosphate dehydrogenase (GAPDH) probe was derived by reverse tran-

scriptase-PCR of Raji mRNA using the following primen: S’GGG- CGCCTGGTCACCAGGGCTG3‘ and S‘GGGGCCATCCACAGT- CTTCTG3’.

Wesfern blotting. Nuclei were prepared according to the method used by Xie et al.?‘ and whole-cell lysates were prepared according to the method used by Sambrook et al.” Proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and then blotted onto nitrocellulose. After blocking with 2.5% nonfat dry milk solution, the filter was incubated with anti-LYSP100 antibodies at 1 pgl mL. Bound antibody was detected with the ECL system (Amer- sham).

Antibodies to LYSPlOO und SP100. LYSPIOO and SPlOO were expressed in bacteria using the pGEX2tk vector (Pharmacia. Piscata- way, NJ). For LYSPIOO, DNA corresponding to amino acids 306 to 412 of the A form was amplified by PCR from the LYSP100-A cDNA using the following oligonucleotides: ( I ) sense. 5’-AAA- AAAGGATCCAAGTCACTAATGAAGGAGAACC-3’, and (2) antisense, S’-AAAAAAGAA’ITCGGAGGATCAGGCAGAAAATCC- 3’. For SPIOO, DNA corresponding to amino acids 333 to 480 were obtained by reverse transcriptase-PCR using Raji mRNA and the following oligonucleotides ( I ) sense. S’-AAAAAAGGATCCAGG- TCTGGCCTCCAACTAAGTCTT-3’. and (2) antisense. S’-AAA-

products were digested with ButnHl plus EcoRl before cloning. An internal BumHI site in the SPlOO gene (at amino acid 333) was used for glutathione-S-transferase (GST) cloning. GST alone, GST- SP100, and GST-LYSP100-A proteins were purified by standard procedures.” Female New Zealand white rabbits were immunized with a primary boost of 500 pg soluble GST-fusion protein, followed by an initial boost with 200 pg protein 4 weeks later plus subsequent boosts every 2 weeks also with 200 pg protein. Blood samples were taken 12 days after every boost. GST alone and GST-fusion proteins were coupled to Affigel (BioRad. Richmond, CA) according to the manufacturer’s specifications and used for affinity purification of LYSPIOO and SPl00 antibodies. Briefly, antiserum was diluted 1:s in 10 mmol/L Tris (pH 7.5) buffer and passed several times over a GST-alone Affigel column to remove anti-GST Specificities. Diluted antiserum was next passed over the respective GST-fusion protein column several times and washed extensively with Tris buffer. Anti- bodies bound to the column were eluted with 1 0 0 mmollL glycine (pH 2.5) and then neutralized with 2 mol/L Tris (pH 7.5). Polyclonal mouse anti-SP100 antiserum was generated against a maltose-binding protein-SP100-fusion protein ah previously described.”’

1nzmunojhtore.scent cell b mining and conjbcul rnicro.scop,v. The Epstein-Barr virus-transformed human B lymphoblastoid cell line, MBB I , was obtained from Dr D. Allman (National Cancer Institute). Magnetic bead-purified (Dynal Corp, Lake Success, NY) CD19’ peripheral blood B lymphocytes were obtained from Drs T. Selvaggi and W. Strober (National Institute of Allergy and Infectious Dis- eases). Cells were attached to glass cover slips coated with Conca- navalin A (Sigma, St Louis, MO) before fixation with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes at 25°C. Cells were permeabilized with 0.5% Triton X-IO0 i n PBS for 5 minutes at 25°C. All antibody staining was performed at 25°C. Rabbit antibodies were used at 1 to 2 pg/mL diluted i n PBS + 5 % fetal calf serum + 0.1% sodium azide. The mouse anti-PML antibody, 5E10.“ was obtained from Dr R. van Driel (University of Amster- dam, the Netherlands) as a tissue culture supernatant. Mouse anti- SplOO serum was used at a 1: lOO dilution. Fluorescein isothiocya- nate-coupled donkey anti-rabbit I& (minus antimouse specificities: Jackson Immunoresearch, West Grove. PA) and Texas Red-coupled donkey anti-mouse Ig (minus antirabbit specificities; Jackson Immu- noresearch) were used at I O yg/mL. Stained cells were analyzed using a BioRad MRC 600 confocal laser scanning microscope and

AAAGAATTCTCCTGACATTCTGCAGGCCAAGTC-3’. PCR




NOVEL NUCLEAR STRUCTURES DEFINED BY LVSPlOO

BioRad Comos software. Confocal images were merged using Adobe Photoshop 3.0 (Adobe Corp. Mountain View, CA).

Tmn.$ection of HeLa cells. A mammalian expression vector for LYSP100-B was created by cloning LYSP100-B cDNA into the vector pCGN” as follows. LYSPlOO-B cDNA was amplified by PCR using the following primers: S’TIITITGGTACCTGGCCC- AGCAGGGCCAGCAGG3’ and 5’TTfTTTGGATCCTATTTT- AGGGTGCCATTTGCTGAAT3’. The PCR product was digested with Kpnl and BomHI and ligated into the same sites in pCGN. For transfection, HeLa cells were plated at 25% confluency on glass cover slips in the wells of a 24-well plate and grown in Dulbecco‘s modified essential medium supplemented with 10% fetal calf serum. Cells were transfected with the CapoI procedure” using 1 pg pCGN- LYSP100-B DNA per well. After addition of DNA, cells were incubated for 16 hours at 37°C and then placed in fresh media. Cells were grown on the cover slips for an additional 24 hours before staining with anti-LYSP100 antibodies, as already described.

Immuno-EM. MBBl cell cultures were fixed for 1 hour at 4°C with 4% formaldehyde in 0.1 molL phosphate buffer (pH 7.3). During fixation, the cells were scraped from the plastic substratum and centrifuged. Pellets of fixed material were dehydrated in increas- ing concentrations of methanol and embedded in Lowicryl K4M (Chemische Werke Lowi, Waldkraiburg, Germany) at low tempera- ture as previously described.’* Polymerization was performed for 5 days at -30°C under long-wavelength UV light. Formvar carbon- coated copper grids (mesh 200) bearing ultrathin sections of Lowi- cryl-embedded material were stained for immunogold detection by first incubating in a bovine serum albumin solution for 30 minutes to suppress nonspecific binding. Grids were then floated on IO-pL drops of an LYSPl00 antibody solution diluted to 10 pg/mL in PBS for 2 hours at 25°C. After a IS-minute wash in PBS, the grids were floated for 30 minutes on a 10-pL drop of a 1:25 dilution of goat anti- rabbit IgG conjugated to gold particles 10 nm in diameter (Biocell Research Laboratories, Cardiff, UK). For double staining, uncoated grids (mesh 300) were first stained for LYSPl00 on one side as already described, and then the opposite sides of the grids were stained similarly for PML using 15-nm gold particle-conjugated anti-rabbit IgG. As a specificity control for the immunogold labeling,

1425

all steps were performed normally but the primary antibody was omitted.

RESULTS

LYSPIOO gene cltaracterimtion. In an effort to identify novel lymphoid-restricted transcription factors, we combined subtractive cDNA methodology with random automated DNA sequencing. A subtracted cDNA library was prepared that was enriched for genes expressed in the Burkitt’s lymphoma cell line, Raji, and not in the erythroleukemia cell line, K562.” One subtracted cDNA clone was chosen for study by virtue of its homology with an autoantigen, SP100,’h.‘7 that had been previously localized to PML NB subnuclear domains.’ Northern blot analysis showed that this gene was expressed as three major mRNA transcripts. 0.9. 1.6, and 3.0 kb in length, that were detected in all three lymphoid tissues examined (spleen, thymus, and tonsil) but not in three nonlymphoid tissues (Fig 1). LYSPlOO mRNA was expressed in all mature B-lymphocyte and plasma cell lines tested (Fig 1, and data not shown), but it was only variably expressed in T-lymphocyte lines and was not detectable in several nonlymphoid cell lines (Fig 1 ) . Expression of the gene is thus lymphoid-restricted both in human tissues and in cell lines. Consequently, we named this gene LYSPIOO, for lymphoid-restricted homologue of SPIOO. By contrast, SPlOO expression is widespread’.’“’’ (and A.L. Dent, unpublished results, January 1994).

To isolate full-length LYSPlOO cDNAs, a Raji cDNA library was screened with the partial LYSPlOO cDNA, and two cDNA clones, both 3.0 kb long, were chosen for further analysis (Fig 2A). The sequences of LYSPIOO-A and LYSPIOO-B cDNAs have identical 5’ and 3‘ sequences but differ by two short insertions in the middle of the LYSPIOO- B cDNA, which probably result from alternative splicing.

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Fig 1. Expression of LYSPlOO mRNA in human lymphoid cell lines and tissues. A Northern blot was prepared using 2 p g poly(A)+ mRNA from human cell lines (lanes 1 to 11) or tissues (lanes 12 to 17) and hybridized with a radiolabeled LYSPlOO probe or a GADPH probe. Cell lines: Hela, cervical carcinoma; K562, myeloerythroleukemia; Hut78 and Jurkat, T cell; Nalm6, pre-B cell; and Raji, NAB, MBB1, VDSO, WIL2-NS, and ARH77, mature B cell.

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1426 DENT ET AL

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LYsP100 / sP100 TTC d o m a i n Homology Regions Domain

Fig 2. Amino acid sequence of LYSPlOO and comparison to SP100. (AI Two LYSPlOO protein isoforms aligned with 2 SPlOO protein isoforms and HNPPl (Genbank accession no. L223431. Identical amino acids are in black and conservative amino acid changes in grey. (B1 Schematic representation of regions of high sequence homology between LYSPlOO isoforms, SPlOO isoforms, and HNPP1. Striped boxes indicate regions in LYSPlOO, SPlOO, and HNPP with high amino acid similarity. Additional regions of similarity within these proteins include the PHD/TTC domain (B21 and the bromodomain (checkered box).

Other LYSPIOO cDNAs account for the 0.9- and 1.6-kb mRNAs and are also most likely the products of alternative splicing (A.L.D., data not shown). The insertions in the LYSPIOO-B cDNA alter the size of the predicted open reading frame: LYSPlOO-A encodes a protein of 412 amino acids with a molecular weight of 46 kD, whereas LYSP100-B encodes a protein of 882 amino acids with a molecular weight of 100 kD. The amino-terminal 158 residues of both LYSPlOO protein isoforms have 61% amino acid homology

with the amino-terminal region of a previously described SPlOO cDNA isoform'" (termed SP100-A in Fig 2A and B). Interestingly, the additional 400 amino acids found in LYSPIOO-B but not in LYSPIOO-A showed significant homology with another previously cloned protein, HNPP (Fig 2A and B). HNPP is highly inducible by interferon but has no known function.3'

To identify potential SPlOO cDNA isoforms that encoded a HNPP domain, we screened a HeLa cDNA library using a




NOVEL NUCLEAR STRUCTURES DEFINED BY LYSPlOO 1427

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Fig 2. (cont'd). (C) Alignments of the PHDmC domain and bromodomain of LYSPlOO-B with other PHDmC- andlor bromodomain- containing proteins from the database. The spacer region between PHDllTC domains and brornodornains of LYSPlOO-B and TlFl are also aligned to show the similarity in this region between the 2 proteins. Identical amino acids and highly conservative amino acid substitutions compared with LYSPlOO-B are in black. References or accession numbers for the protein sequences: TIF1"; MLL, 003164(SP); trx, P206591SP); BR140, M91585(GBI; hCBP, S39162(PIR); mCBP, S39161(PIR); p300, U01877(GB); dFSH, P13709(SP); hFSH, X62083(GB); ORFX, D26362(GB); BDF1, 218944(GB); GCN5, Q03330(SP); CCG1, P21675(SP); SPT7, L22537(GB); BRAHMA, P25439(SP); hBRG1, S669101GB); hBRM, X72889(GB); SNFZ, P22082(SP); STH1, M83755(GB); and PB1, X908491GB). SP, Swiss protein; GB, GenBank.

probe derived from the LYSPIOO-B HNPP homology region. Based on the sequence of a novel HNPP-like clone isolated from this screen, PCR primers were designed that were used with SPlOO primers to amplify a novel SPIOO isoform from Raji cDNA. Sequencing of a full-length cDNA for this SPIOO isoform, termed SP100-B, revealed that it is identical to SP100-A over the first 477 amino acids, but it has a carboxy-terminal extension that is highly similar to but shorter than the carboxy-terminal domain of LYSP100-B (Fig 2A and B). Thus, the LYSP100-B and SP100-B isoforms share amino acid similarity throughout the length of the predicted proteins (Fig 2B), suggesting that these two proteins had a common evolutionary precursor.

Further sequence analysis of the LYSPIOO-B isoform revealed two additional domains found in a variety of transcriptional regulatory proteins: a PHDlTTC d~main"".~' and a bromodomain'" (Fig 2B and C). The PHDlTTC domain is a cysteine-rich motif with the general structure, C4HC3, that is presumed to coordinate zinc. Interestingly, like LYSPIOO-

B, several coactivator proteins contain both a PHD/TTC domain and a bromodomain, including TIF1,43 CREB binding factor,JJ and ~300:~ The PHD/TTC domain and bromodomain of LYSPIOO-B are most closely related to those of TlFl , a potential transcriptional coactivator for nuclear hormone receptor^."^ In addition, the spacer regions between PHD/TTC domains and bromodomains of LYSPIOO-B and TlFl are similar in length and have homology to each other (Fig 2C), suggesting that the carboxy termini of these two proteins may have related functions. Taken together, the presence of the PHD/TTC domain and bromodomain in LYSPIOO-B suggests that LYSPIOO-B may be involved in transcriptional regulation.

Subcellular localization qf LYSPlOO protein. We next generated affinity-purified rabbit antibodies against LYSPIOO to determine if LYSP100, like SPIOO, was localized in PML NBs. The anti-LYSP100 antibodies were raised against the carboxy terminus of LYSP100-A, since this region displayed little homology to SPIOO. These anti-




142% DENT ET AL

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Nuclear Whole Cell Extract Extract h h

-100 kD

-69 kD

-46 kD

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1 2 3 4 5 Fig 3. Western blot analysis of LYSPlOO protein. Nuclear or

whole-cell extracts were prepared from B-cell lines expressing LYSPlOO mRNA (ARH77 and Raji) or from cell lines lacking LYSPlOO mRNA (K562 and Reh) and analyzed by immunoblotting with affinity- purified anti-LYSP100 antibodies. Arrows indicate multiple LYSPlOO protein isoforms.

LYSPlOO antibodies should also recognize the LYSP100-B isoform, since most of the LYSPIOO-A carboxy terminus is shared by LYSPIOO-B (Fig 2A and B). Specificity of the anti-LYSP100 antibodies was tested by Western blot analysis of nuclear and whole-cell extracts (Fig 3). A set of protein isoforms was observed in cells that express LYSPlOO mRNA (ARH77 and Raji), but not in cells lacking LYSPlOO mRNA (K562 and REH). The LYSPlOO isoforms ranged in size from 65 to 150 kD and may represent the products of alternatively spliced LYSPlOO mRNAs or may include proteolytic degradation products. In vitro translation of LYSPIOO-A and LYSP100-B isoforms showed that the protein products mi- grate more slowly in sodium dodecyl sulfate gels than predicted by the DNA sequence, as previously observed for SP10026 (and A.L.D., data not shown). By this criterion, LYSPlOO-A mRNA can account for the 65-kD LYSPlOO protein isoform seen on Western blots, and LYSP100-B mRNA can account for the 150-kD LYSPIOO protein isoform. In addition, the 100-kD LYSPlOO protein appears to be encoded by another LSYPlOO cDNA isoform (A.L.D., data not shown). The lower-molecular-weight bands on the Western blots were detected in all cells regardless of LYSPIOO mRNA levels, and therefore represent cross-reacting protein species (Fig 3).

Next, subcellular localization of LYSPlOO was investi- gated using the affinity-purified anti-LYSP100 antibodies for immunofluorescent staining and confocal laser microscopy of the MBBl human lymphoblastoid cell line and human peripheral blood B lymphocytes (Fig 4). LYSPlOO localized to five to 20 dot-like structures in the nucleus of MBB 1 cells (Fig 4A, D, and G). LYSPIOO-associated dots were also detected in nontransformed human peripheral blood B cells (Fig 41,3 cells shown). On average, the number of LYSPIOO- associated dots was significantly lower in peripheral blood

B cells than in the Epstein-Barr virus-transformed cell line: one to three structures were seen per peripheral blood B cell, with some cells having no detectable dots. LYSPlOO was detected in nuclear dots regardless of whether the cells were fixed with paraformaldehyde, methanol, or acetone, and nuclear localization of LYSPlOO dots was confirmed by costaining the cells with a dye specific for DNA (data not shown).

Specificity of LYSPIOO staining was demonstrated in two ways. First, the characteristic LYSPlOO nuclear dots were found only in LYSPlOO-expressing cells (MBB1, VDSO, and ARH77), and not in cells that are negative for LYSPlOO mRNA (K562 and HeLa) (A.L.D., data not shown). As a second test of antibody specificity, HeLa cells were transfected with a eukaryotic expression vector for LYSPlOO-B and then costained with the anti-LYSP100 antibodies (Fig 5A, green staining) and with .the 5E10 monoclonal anti-Ph4L antibody (Fig 5A, red staining). Ten percent to 20% of the cells showed a dot-like nuclear staining with anti-LYSP100 antibodies that was similar to the LYSPlOO staining observed in MBBl lymphoblastoid cells. Nuclear localization of LYSPlOO dots was confirmed by Nomarski differential-interference contrast microscopy (Fig 5B). The remainder of the cells, which presumably did not incorporate DNA during the transfection, did not show this LYSPlOO staining pattern. These specificity studies demonstrate that although anti-LYSP100 antibodies detected minor cross-reacting protein species in all cell types by Western blot analysis, these cross-reacting proteins were not readily detectable in the immunofluorescence assay. Furthermore, the HeLa transfection experiment demonstrates that LYSPlOO nuclear structures can be generated in nonlymphoid cells and thus do not require other lymphoid-specific proteins for their for- mation.

Surprisingly, in transfected HeLa cells, LYSPlOO and PML staining patterns were largely nonoverlapping (Fig 5A). Similarly, the majority of LYSPIOO and PML nuclear structures in MBBl and peripheral blood B cells were not coincident (Fig 4C, F, I, and L). In contrast, SPlOO and PML colocalized precisely in MBBl cells in nuclear structures with the characteristic appearance of PML NBs (Fig 4M to 0), confirming previous results.' We next tested whether the LYSPlOO nuclear dots coincided with other previously described subnuclear domains. LYSPlOO immunofluorescent staining was entirely nonoverlapping with the nuclear speckles detected with antibodies to the splicing factor, SC35,46 and the nuclear dots detected with antibodies to p80- coilin4' (data not shown). Thus, LYSPlOO was localized to subnuclear structures that are distinguishable from PML NBs, interchromatin granules and interchromatin fibrils (containing SC35), and coiled bodies (containing p80-coilin).

Interestingly, in approximately 5% of the MBBl cells, PML dots and LYSPlOO dots exactly coincided within a confocal optical slice (Fig 4C and I). In some cells, only one dot costained for LYSPlOO and PML (Fig 4C), whereas in other cells LYSPlOO and PML coincided in more than one structure (Fig 41). In some cells, the pattern of PML/ LYSPIOO colocalization was also striking, in that the





LYSPIOO PML MERGE

Fig 4. Laser-scanning confocal microscopy of cells immuno- fluorescently stained with antibodies to LYSPlOO andlor PML (A to L) or with antibodies t o SPlOO andlor PML (M to 01. A to I and M t o 0 were derived from the human lymphoblastoid cell line, MBB1, with each row repre- senting images obtained from a single MBBl cell. J to L were derived from human peripheral blood B lymphocytes. Three representative cells are shown in each panel. Green fluorescence lLYSPl00 or SPlOOl and red fluorescence (PML) were collected simultaneously and then separated and merged digitally. Colo- calization of green and red fluorescence yields a yellow image.




Fig 6. Laser-scanning confocal fluorescent microscopy z-series of a MBBl cell stained with anti-LYSP100-B antibody (green fluorescence1 and anti-PML antibody (red fluorescence). Twenty optical slices along the z-axis are shown.





Fig 5. (A) Laser-scanning confocal fluorescent microscopy of HaLa cells transfected with a LYSPlOO-B expression vector and then stained with anti-LYSP100-B antibody (green fluorescence1 and anti-PML antibody (red fluorescencel. At left is a binucleated transfected cell, and at right are several nontrandected calls. (B) Nomarski differential-interference contrast microscopy of the cells in A.

LYSPlOO immunofluorescence appeared to be surrounded by a ring of PML immunofluorescence (Fig 41). Based on an analysis of a z-series of 20 confocal optical slices through this cell (Fig 6), PML appeared to form a shell around a central LYSPlOO core. This observation rules out the possibility that colocalization of LYSPlOO and PML in these structures occurred simply by chance superimposition.

Next, the relationship of SPlOO dots to LYSPlOO dots was judged by costaining with a polyclonal mouse anti- SPlOO antibody and the polyclonal rabbit anti-LYSP100 antibody (Fig 7). In most cells, SPlOO and LYSPlOO were detected in nuclear dots that did not overlap (data not shown). However, in some cells dot-like structures containing both SPlOO and LYSPlOO were detected, and often a single nucleus contained several such structures (Fig 7). Approximately the same minor fraction of MBBl cells (-5%) showed LYSPlOO/SPlOO colocalization as showed LYSPlOOPML colocalization. Thus, in a minority of cells, LYSPlOO colocalized with PML or SP100. In some images, the colocalization was reminiscent of PML NBs (Fig 41). However, a more precise definition of LYSP100-associated nuclear structures required the higher resolution afforded by immuno-EM.

Immuno-EM characterization of LANDs. We used immuno-EM to visualize the ultrastructural morphology and subcellular localization of LYSP100-associated structures. After fixation and embedding of MBBl lymphoblastoid cells, thin sections were reacted with LYSPlOO antibodies alone (Fig 8), with both LYSPlOO and PML antibodies (Fig 9), or with both LYSPlOO and SPlOO antibodies (data not shown). LYSPlOO antibodies were secondarily reacted with anti-rabbit IgG antibodies coupled to IO-nm gold particles, whereas PML or SPlOO antibodies were detected with antibodies coupled to 15-nm gold particles. LYSPlOO antibodies decorated electron-dense structures that were round to irreg- ular in shape. These structures ranged from 0.1 to 0.3 pm in diameter, and thus were smaller than PML NBs, which were 0.3 to 1 .O pm. The electron density of LYSP100-associated structures was often uneven, and in many instances they appeared to have a central core that was more electron-dense (Figs 8A and 9A). Specificity of LYSPlOO immunogold detection was checked in two ways. First, the characteristic electron-dense structures were not observed when the gold- labeled goat anti-rabbit IgG alone was used for detection. Second, electron-dense structures detected by anti-LYSP100 antibodies were observed frequently in MBB 1 cells but only rarely in HeLa cells, as predicted by the preferential expression of LYSPlOO in lymphoid cells.

LYSP100-associated structures and PML NBs were morphologically distinct in several respects. First, LYSPlOO structures often appeared more electron-dense than PML NBs (Fig 9C and D, LYSPlOO structures marked by big arrows-PML NBs marked by small arrows). Second, LYSPlOO was never detected in a ring-like electron-dense structure, which is a hallmark of PML NBs.**~ Third, the dot-like LYSPlOO structures were uniformly decorated by LYSPlOO antibodies (Fig 8A to D), whereas PML tended to localize over the outer-ring structure of PML NBs (Fig 9C and D). LYSPlOO therefore appears to be associated with a novel nuclear domain that is distinct from PML NBs and other well-characterized subnuclear structures. We therefore propose to name these subnuclear domains “LANDs,” for LYSP100-associated nuclear domains.

In sections stained with both LYSPlOO and PML antibodies, most LANDs were found to stain only with LYSPlOO. Furthermore, most PML staining was detected in typical PML NBs. However, some LANDs were decorated with both 10-nm and 15-nm gold particles, indicating the presence of both LYSPlOO and PML, respectively (Fig 9). These PML and LYSPlOO costaining structures appeared to have the characteristics of LANDs, as already described, rather than the characteristics of PML NBs. Indeed, LANDs that contained PML could not be readily distinguished by morphology from those that did not. The ratio of LYSPlOO to PML gold particles in the structures shown in Fig 9 is 2.0 -C 0.49. The relatively low variation in this ratio points to a consistency in the molecular composition of the LANDs to which both LYSPlOO and PML localize. In contrast to LYSPlOO, antibodies to SPlOO stained characteristic PML NBs exclusively, and were not detected in structures with LAND morphology (data not shown).

EM provided several intriguing refinements for the characterization of subcellular distribution of LANDs. Most LANDs were observed within the nucleoplasm in the interchromatin space (Figs 8A and 9A and D). However, many LANDs were localized precisely at the nuclear envelope, sometimes on the nuclear face (Fig 9C) and in other cases on the cytoplasmic face (Figs 8B and C and 9B). Frequently, these LANDs were seen at the tip of deep invaginations of the nuclear envelope into the nucleus (Figs 8B and 9B). Approximately one fifth of the LANDs were observed in the cytoplasm and were morphologically indistinguishable from LANDs present in the nucleoplasm (Fig 8D). LANDs that contained PML showed a similar subcellular distribution compared with LANDs devoid of PML. In summary, morphologically similar LANDs were observed in the nucleo-

4

Fig 7. Lasar-scanning confocal microscopy of MBBl cells stained with anti-LYSP100 (green fluorescence) and anti-SP100 antibodies (red fluorescence). colocalization of green and red fluorescence yields a yellow image. A, B, and C, 3 individual cells representative of the minority of cells where LYSPlOO and SPlOO colocalized.




1432 DENT ET AL

plasm, at the nuclear membrane, and in the cytoplasm, rais- ing the possibility that LANDs are transported between these compartments.

DISCUSSION

We have cloned a novel lymphoid-restricted nuclear factor, LYSPlOO, that is homologous to SP100, a protein found in dot-like nuclear domains termed PML NBs. As analyzed by confocal immunofluorescence microscopy, LYSPlOO was also localized in dot-like subnuclear structures, but, surprisingly, LYSPlOO dots were generally nonoverlapping with PML NBs. By immuno-EM, LYSPlOO was found in novel electron-dense globular structures that were morphologically distinct from PML NBs and other previously described subnuclear structures. Thus, LYSPlOO molecularly defines a novel subnuclear domain, which we term LAND, for LYSP100-associated nuclear domain. interestingly, in a mi- nor population of LANDs, LYSPlOO and PML did colocal- ize, as judged by immuno-EM. Our data thus reveal LANDs as a new class of nuclear structures associated with PML.

LYSPlOO and SPlOO genes likely evolved from a common ancestor, since they encode proteins that share several regions of homology. All LYSPl00 and SPlOO isoforms share a highly conserved 157-amino acid amino-terminal domain. In addition, one isoform of LYSPlOO, LYSPlOO-B, contains a carboxy-terminal extension of 470 amino acids that bears homology with HNPP, a nuclear phosphoprotein that is strongly induced following interferon treatment of cells.3y We used this LYSPlOO isoform to identify a previously undescribed SPlOO isoform, SP100-B, which also contains a carboxy-terminal HNPP domain. A common feature of SP100, HNPP, and PML is that they are strongly upregulated by interferon treatment of cells.48349 By contrast, interferon treatment has only a modest (-twofold) effect on LYSPlOO mRNA expression (A.L. Dent, unpublished observations, September 1993).

Of particular note, LYSP100-B shares two homology regions with several transcriptional regulatory proteins, the PHD/TTC domain and the bromodomain. The PHD/TTC domain is defined by a characteristic arrangement of cysteine and histidine residues, C4HC3,40.4’ and is a potential zinc- coordination motif.50 Two PHD/TTC domain proteins, CBP and p300, mediate transcriptional activation through the CAMP-responsive transcription factor, CREB.s1.52 The PHD/ TTC motif is also repeated four times in the Drosophila homeotic protein, trithorax, a positive regulator of transcription that is responsible for maintenance of the transcription of homeotic selector genes5’ The MLL gene, a trithorax homologue translocated in a variety of human leukemia^:^,'^ has particularly high sequence homology with trithorax in the PHD/TTC region.

The closest homologue to LYSP100-B in the PHD/TTC domain is TiF1, a potential coactivator protein for nuclear hormone receptors.43 TIFl binds to the AF-2 activation domains of nuclear hormone receptors in a ligand-dependent fashion and can stimulate transcription through these receptors in yea~t .4~ Interestingly, TIF- 1 displays a more extended homology with LYSP100-B that includes the bromodomain and the spacer region between the two domains, suggesting

that these two domains perform related functions in the two proteins. In addition to TiF1, the coactivator proteins, CBP and p300, also contain both a bromodomain and a PHD/ TTC domain. The bromodomain is also found in CCG l / hTAFI1250,5”sh a protein that is tightly associated with TATA binding factor and is required for the function of certain transcriptional activation domain^.^' A large, functionally distinct class of bromodomain proteins are homo- logues of the Drosophila homeotic protein, Brahma, and the yeast SWIZ/SNF2 protein, and appear to contribute to a multiprotein complex that disrupts ~hromatin.~’ Although the precise function of the bromodomain is not yet clear, deletion of this domain in the yeast transcriptional adaptor protein, GCNS, impairs its ability to activate transcription mediated by certain activation domains.” These sequence homologies provide provocative, albeit preliminary, indications that LYSPlOO may participate in transcriptional regulation. The lymphoid-restricted expression of LYSPl00 further suggests that LYSPlOO does not perform a merely “housekeeping” function, but instead may act to regulate transcription in a cell type-specific fashion.

LANDs were distinct in location and morphology from other subnuclear structures that have been molecularly defined, including interchromatin granules, perichromatin fibrils, coiled bodies, and PML NBs. EM showed LANDs to be round or irregularly shaped electron-dense structures ranging in size from 0. I to 0.3 pm. Three-dimensional confocal image plane reconstruction of LANDs showed them to be solid, roughly spherical structures (A.L.D., J.Y ., and L.M.S.. unpublished data, March 1994). Thus, the morphology of LANDs as revealed by immunofluorescence or EM is not notably distinctive, and consequently, this novel nuclear sub- domain could not have been appreciated without the cloning and characterization of LYSP100. At present, since LYSPlOO is a lymphoid-restricted protein, it is not possible to convincingly demonstrate that LANDs exist in nonlymphoid cells. However, in this regard, it is noteworthy that when LYSP100-B was expressed in HeLa cells by transfection, LYSPlOO antibodies detected dot-like nuclear structures by immunofluorescence that were indistinguishable from LANDs detected in lymphoid cells. One interpretation of this result is that the ectopically expressed LYSPlOO was incorporated into LAND-like structures that preexisted in HeLa cells. Alternatively but seemingly less likely, LYSPlOO might organize de novo LANDs within HeLa cells.

The ultrastructural location of LANDs may provide some intimation of their function. The greatest number of LANDs was observed in the nucleoplasm, where they could be detected in the interchromatin regions (Figs 7 and 8) and in the nucleolar regions (data not shown). However, in addition, many LANDs were directly touching the nuclear envelope, both on the nuclear and cytoplasmic face, and approximately one fifth of the LANDs were in the cytoplasm proper. In- triguingly, LANDs were frequently observed at the tip of deep invaginations of the nuclear membrane (Figs 7B and 8B). It is unclear how such invaginations in the nuclear envelope are formed and maintained, but they could conceivably represent functional specializations of the nuclear enve-




Fig 8. Immuno-EM of MBE1 lymphoblastoid cells stained with anti-LYSP100 antibodies. LANDs were detected in the nucleoplasm (A), at the nuclear envelope (B and C), and in the cytoplasm (D). Nu, nucleolus; NE, nuclear envelope; M, mitochondrion. Original magnification: A, x 66.000; B to D, x 39,000.

lope. These observations suggest the possibility that LANDs may traverse the nucleus to the cytoplasm, or vice versa. In the former case, LANDs might be multisubunit structures that, like ribosomes, are assembled in the nucleus but function in the cytoplasm. Alternatively, LANDs might be directly involved in the transport of macromolecules such as RNA from the nucleus to the cytoplasm. On the other hand, LANDs, like snRNPs," may be assembled in the cytoplasm and then imported into the nucleus. Once in the nucleus, LANDs may serve as sites where specialized nuclear functions take place, or they may be storage areas for proteins that can be released from LANDs to function elsewhere in the nucleus.

Finally, our data provide evidence for the association of PML with a subnuclear structure other than the PML NB. PML-containing LANDs were found in the nucleoplasm, at the nuclear membrane, and in the cytoplasm,

and were morphologically indistinguishable from LANDs lacking PML. Previously, PML has been localized by immunofluorescence to the cytoplasm of some cells,'' yet PML NBs have only been described by EM in the nucleoplasm. Therefore, some of the previously described cytoplasmic PML may be attributable to LANDs. Furthermore, although the majority of PML in the nucleus is associated with PML NBs. some of the nuclear dots detected by immunofluorescence with anti-PML antibodies could represent LANDs. Recent studies have demonstrated that PML can suppress the growth and malignant transformation of cells,""' and can repress the activity of certain promoters in transient transfection assays.'3 The present results suggest that LANDs should be taken into consider- ation in models of PML function.

One possibility raised by the presence of PML in both LANDs and PML NBs is that LANDs might be precursors in




1434 DENT ET AL

Fig 9. Simultaneous visualization of LYSPlOO and PML in MBBl lymphoblastoid cells by immuno-EM. Anti-LYSP100 antibodies were detected with antibodies coupled with 10-nm gold particles, and anti-PML antibodies with antibodies coupled with 15-nm gold particles. LANDs containing both LYSPlOO and PML were detected in the nucleoplasm (A and D) and at the nuclear envelope (B and C). C and D, large arrows point to LANDs and small arrows to PML NB structures stained only with anti-PML antibodies. Nu, nucleolus; NE, nuclear envelope. Original magnification: A, B, and D, x 51,000; C, x 42,000.

the biogenesis of PML NBs. Conceivably, PML-containing LANDs might coalesce to generate the ring-like PML NBs. In this regard, the confocal immunofluorescence image of LYSPIOO surrounded by a ring of PML (Fig 41) is especially intriguing. Given the annular nature of PML NBs by EM, it seems possible that the ring-like structures in Fig 41 are unusu- ally large PML NBs. Thus, in a small percentage (4%) of cells, LYSPIOO may be located in PML NBs. By immuno- EM, we were unable to detect LYSPIOO in characteristic PML NBs, but this failure may simply reflect the difficulty of de- tecting rare events by EM. If LYSPIOO is indeed present in a small subpopulation of PML NBs, it seems possible that LANDs are precursors to these structures.

ACKNOWLEDGMENT

We thank Drs T. Selvaggi and W. Strober for providing purified human peripheral blood B lymphocytes, and J. Powell for assisting

with Fig 2. The SEI0 anti-PML monoclonal antibody was kindly provided by Drs L. De Jong and R. Van Driel. We also thank T. Tran for expert technical assistance.

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1996 88: 1423-1426

AL Dent, J Yewdell, F Puvion-Dutilleul, MH Koken, H de The and LM Staudt bodiesclass of subnuclear structures and their relationship to PML nuclear LYSP100-associated nuclear domains (LANDs): description of a new

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LYSP100-Associated Nuclear Domains (LANDs - Blood

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