Insulation of the Chicken β-Globin Chromosomal Domain from a Chromatin-Condensing Protein, MENT

Abstract

Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken ␤-globin domain. We observed two sharp transitions of MENT concentration coinciding with the ␤-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation.In eukaryotic cells, the DNA is repeatedly wrapped around histone octamers to form nucleosomes. Nucleosome arrays are packed through a hierarchy of folding levels into higher-order chromatin structures with a variable degree of condensation (for reviews, see references 32, 67, 68, and 70). Chromatin condensation is especially tight in heterochromatin, representing the repressed fraction of genetic material (30, 57). Heterochromatin becomes very abundant in the nuclei of terminally differentiated cells, where the majority of formerly active genes are repressed and condensed (20,21). In this paper we investigate how the unique properties of heterochromatin in terminally differentiated cells integrate the general mechanism of gene regulation by histone modifications.One of the most intriguing aspects in the process of heterochromatin formation is that chromatin condensation does not spread to the actively transcribed tissue-specific genes, which still maintain an open, nuclease-sensitive conformation (66). For example, in the chicken ␤-globin domain, a 30-kb region of active DNase-sensitive chromatin is separated from repressed and condensed chromatin by gene boundary elements (2, 11). The repressed chromatin is associated with a special pattern of histone modification, most notably a decreased level of histone H3 and H4 acetylation (28, 43) and an increased level of histone H3 methylated at lysine 9 (H3meK9) (42). The H3meK9 binds to the chromodomain of heterochromatin protein 1 (HP1) (49), and this association has been proposed to determine the repressed state of heteroc...

Dynamic relocation of epigenetic chromatin markers reveals an active role of constitutive heterochromatin in the transition from proliferation to quiescence

“…Cell suspensions in LIS were filtered through gauze, centrifuged for 10 minutes at 200 g, 4°C and resuspended in RPMI-10/HEPES at 4×10 7 cells/ml. Nuclear isolation and western analysis Isolation of nuclei from mouse lymphocytes and cultured NIH/3T3 cells, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), western blotting and ECL detection were performed as described (Istomina et al, 2003). For detection of HP1 and histones, proteins were transferred to Immobilon-P PVDF membranes (Millipore) in 20% methanol.…”

“…NIH/3T3 cells were grown on cover glasses, fixed, and probed first with primary antibodies against macroH2A, HP1α, Hp1β, H3K9Me 3 , H3K9Me 2 , and H4K12Ac, followed by secondary antibodies labeled with Alexa Fluor 488 and/or 594 (Molecular Probes) as described (Istomina et al, 2003). All samples were stained with 0.1 µg/ml Hoechst 33258 in PBS.…”

Heterochromatin?many flavours, common themes

“…The tight packaging of chromatin is also evidenced by the finding that a ''branched chain'' antibody raised against four identical, linked, H3 peptides each dimethylated at H3-K9 is specific for pericentric heterochromatin, whereas an antibody raised against a linear peptide stains chromosomes uniformly. (28) Condensation of heterochromatin can also be aided by proteins such as MENT, which binds specifically to methylated H3-K9 (94) and condensins, which can interact with DNMT3B. (95) …”

Role of Heterochromatin Epigenetic Factors in CML

“…We have successfully developed conditions for chromatin immunoprecipitation (ChIP) for MNEI in combination with H3me3K9 based on the protocol that we formerly used for another nuclear serpin, MENT (11). Immunoprecipitated DNA concentrations were determined using real-time PCR detection (SYBR green detection system) with constitutive heterochromatic DNA probe (α-satellite), a silent control gene probe (β-globin), and probes for the promoter and coding regions of two key genes associated with CML: junB whose inactivation leads to a CML-like myeloproliferative disorder and blast crisis (12) and c-myc whose deregulated expression inhibits terminal differentiation in myeloid cells (13).…”

“…Lei20F/Lei25R and Lei24F/Lei13R (see appendix, Table II (4), unclassified myeloproliferative disorder (7); acute myeloid leukemia (11,13). Nuclear samples were normalized to equal loading of histones before electrophoresis.…”