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First published online 12 September 2007
doi: 10.1242/jcs.012955

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Variant-specific patterns and humoral regulation of HP1 proteins in human cells and tissues

¹ Stem Cell and Chromatin Group, Laboratory of Biology, The University of Ioannina School of Medicine
² Laboratory of Pathology, The University of Ioannina School of Medicine, Dourouti, 45 110 Ioannina, Greece
³ Biomedical Institute of Ioannina (FORTH/BRI), Dourouti, 45 110 Ioannina, Greece

View larger version (135K): [in this window] [in a new window]   Fig. 1. Expression of HP1 and HP1 in various tissues and cell types. (A) Identification of HP1 and HP1 transcripts in human tissues by RT-PCR. Lanes: 1, endometrium; 2, myometrium; 3, mammary glands; 4, lymph nodes, 5, skin; L, a 1 kb DNA ladder. Sizes of amplified products (in bp) are indicated on the right. Beta-actin amplification was used as a control. For more details see Materials and Methods. (B,C) Immunocytochemical detection of HP1 and HP1 in (B) human epidermis, gastric mucosa, mammary gland and prostate, and (C) in mouse cerebellar cortex. Red arrows indicate Purkinje cells expressing both proteins. (D) Detection of the HP1 and HP1 proteins in human liver. Red arrows indicate Kupffer cells and black arrows, hepatocytes. (E) Staining of human liver with control (anti-lamin B and anti-me2K4 histone H3) antibodies. In D and E the two upper panels show hepatic lobules, whereas lower panels show inter-lobular space containing bile ducts.

View larger version (123K): [in this window] [in a new window]   Fig. 2. Distribution of HP1 and HP1 in human lymph nodes. (A) Schematic representation of lymph node structure. (B) Double staining for HP1 or HP1 (brown) and B- (CD20) or T-cell (CD4 and CD8) markers (red). (C) Expression of HP1 and HP1 in primary (pf) and secondary follicles comprising a germinal center (GC) and a mantle zone (mz). The area of primary follicles is indicated by dots. (D) Detection of HP1, HP1, Ki-67 and me2K4-H3 in the B- and T-zone.

View larger version (61K): [in this window] [in a new window]   Fig. 3. Redistribution of HP1 in immortalized B- and T-lymphocytes. (A) Monocultures, co-cultures and IL-2-treated cells doubly-stained with anti-HP1 (green) and anti-HP1 (red) antibodies. Coarse HP1 foci that appear upon co-culture or IL-2 treatment are more obvious in the HUT-78 line. Notice the convoluted nuclei in HUT-78 monocultures, a typical feature of Sezary cells. Bar, 18 µm. (B) Morphometric data showing the proportion of HP1 and HP1 foci in the nucleus of HUT-78 and RPMI-8226 cells under different culture conditions (blue bars: 0-2 µm foci; green bars: 2-4 µm foci and orange bars: 4-6 µm foci). Optical sections and `projections' of 15 different cells were analyzed in each case. (C) Fold-difference in the abundance of the various classes of HP1 particles upon co-culture or IL-2 treatment in relation to controls (monocultures). The results, shown in a tabular form, were derived from the data presented in B. (D) Western blots showing the levels of HP1, HP1 and histone H3 in nuclear extracts of HUT-78 and RPMI-8226 cells before and after co-culture, or treatment with IL-2.

View larger version (52K): [in this window] [in a new window]   Fig. 4. Redistribution of HP1 in peripheral blood lymphocytes (PBLs). (A) Double staining with anti-HP1 (green) and anti-HP1 (red) antibodies. The panel shows non-treated (NT), phytohemagglutinin-treated (PHA), lipopolysaccharide-treated (LPS) and IL-2-treated (+IL-2) human PBLs. Bar, 13 µm. Note the difference in the size of the nucleus. (B) Morphometric data showing the percentage of HP1 and HP1 foci in the nucleus of non-treated and treated cells, as indicated (dark blue bars: 1-2 µm foci; green bars: 2-3 µm foci, orange bars: 3-4 µm foci, and light blue bars: 4-5 µm foci). Optical sections and `projections' of 15 different cells were analyzed in each case. (C) Fold-difference in the abundance of the various classes of HP1 particles upon treatment in relation to controls. The results, shown in a tabular form, were derived from the data presented in B. (D) Western blots of PBLs before and after treatment with LPS or IL-2. The blots were probed for HP1, HP1 and histone H3, as indicated.

View larger version (54K): [in this window] [in a new window]   Fig. 5. Correlation of HP1 redistribution with lymphocyte activation. (A) Co-staining of non-treated (NT) and stimulated (PHA, LPS) PBLs with anti-HP1 (green) and anti-Ki-67 (red) antibodies. Note that cells with coarse HP1 foci are scarce in the control but increase substantially after treatment. As expected, the number of the intensely stained figures is rather small in the LPS sample since B cells constitute a very small percentage (25-28%) of total blood lymphocytes. Bar, 15 µm. (B) Staining of non-treated and stimulated PBLs with anti-HP1 (green) antibodies and BrdU (red). The induced cells have entered the cell cycle and incorporated BrdU. Bar, 15 µm. (C) HUT-78 monocultures and LiCl-treated cells stained with the indicated antibodies. Asterisks denote mitotic cells. Notice that addition of LiCl affects only the rate of cell division and not the distribution pattern of HP1. Bars, 10 µm and 5 µm as indicated. (D) Graphic representation of the mitotic index of HUT-78 cells prior to and after incubation with LiCl. 1,000 cells were analyzed in each case.

View larger version (55K): [in this window] [in a new window]   Fig. 6. Downregulation of HP1 and HP1 during mammalian erythropoiesis. (A) Staining of HMBA-treated mouse erythroleukemia cells (MEL) with anti-HP1 or anti-HP1 antibodies (green) and propidium iodide (red). Arrows indicate differentiated, normoblast-like figures possessing condensed nuclei. Cells with bigger nuclei are undifferentiated early erythroblasts that have not responded to HMBA treatment (for more details see text). Bar, 5 µm. (B) The same experiment as in A using human erythroleukemic cells (K-562). Bar, 5 µm. (C) Immunohistochemical detection of HP1 and HP1 in human bone marrow sections. The images show erythropoietic islands containing early and late erythroblasts (red arrows) stained with the corresponding antibodies and Hematoxylin. (D) Double staining of undifferentiated and partially differentiated mouse eythroleukemia cells (as in A) with anti-HP1 (green) and anti-histone modification (red) antibodies. Note that the anti-me3K9 and anti-me3K27-H3 antibodies readily penetrate the condensed nuclei of the normoblasts, indicating access to intranuclear antigens. Bar, 5 µm.

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