Heterogeneous binding of high mobility group chromosomal proteins to nuclei

Abstract

A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain >90% of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated . The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40% of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction . No histone is observed in the cytosol fraction . Unlike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption . The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them . Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution . On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8% of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35% of the total HMG 1, is stably bound, as is all the HMGs 14 and 17 . The remaining 65% of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption . It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs .Evidence has accumulated that the high mobility group (HMG) chromosomal proteins are required for transcriptionally active chromatin structure . Incubation of chromatin with DNase I preferentially digests transcriptionally active sequences (4,16,34), while selectively releasing the HMGs (15,17,18,33,35) . The preferential DNase I sensitivity of the globin genes in erythrocyte mononucleosomes is abolished by the selective extraction of the nonhistone fraction containing the HMGs, while readdition of purified HMGs restores it (35,36) . The HMGs may also be involved, at least indirectly, in the regulation of differential gene activity during cytodifferentiation since they are themselves developmentally regulated proteins. Cordon and colleagues (7) have shown that the proportions of HMGs 1, 2A, and 2B vary between different chicken tissues and that quantitative changes in HMG proportions are ob-

Activation of Inflammasomes Requires Intracellular Redistribution of the Apoptotic Speck-Like Protein Containing a Caspase Recruitment Domain

“…2 B ). Although the control proteins clearly indicated that the fractionation procedure was highly efficient, ASC was also seen in the cytosol in resting cells, which was not visible by immunofluorescence microscopy and was likely caused by nuclear leakage of the small ASC protein during aqueous fractionation, which is a well-acknowledged problem for small proteins (37, 38). ASC is only 22 kDa in size, and therefore retaining more than 50% of ASC inside the nucleus during fractionation further confirms its predominantly nuclear localization in resting monocytes, and that ASC redistributes from the nucleus to the cytosol following exposure to E. coli total RNA.…”

HMGB1 expression and release by bone cells

“…The UMR 106‐01 cells may represent an intermediate developmental stage between early osteoblast precursors and mature osteoblasts (Iida‐Klein et al, 1992) since they express relatively little, if any, osteocalcin and bone sialoprotein, markers of a mature osteoblast (Lecanda et al, 1998). For example, oocytes of several amphibian species as well as chicken embryonic fibroblasts contain large pools of non‐chromatin‐bound, soluble HMGB1 (Gordon et al, 1981; Kleinschmidt et al, 1983), perhaps analogous to the fivefold increase in UMR HMGB1 levels as compared to the primary osteoblasts. The 100‐fold elevated level of HMGB1 in the media of UMR cells as compared to the MC3T3‐E1 cells cannot be fully accounted for by the fivefold increase in UMR expression of this protein, suggesting a comparatively amplified rate of release in osteoprogenitor cells.…”

High mobility group proteins of amphibian oocytes: a large storage pool of a soluble high mobility group-1-like protein and involvement in transcriptional events.

“…The sizes of the nuclear and cytoplasmic pools of free HMG proteins in cells other than oocytes are not known but the presence of some cytoplasmic (19) is clearly excluded by our findings of relatively large amounts of HMG-A in manually enucleated ooplasms. Probably, the sizes of the specific nucleoplasmic and cytoplasmic pools of HMG proteins will have to be determined for each cell type (17). However, the amphibian oocyte clearly exemplifies the build up of large pools of a soluble HMG-l-like protein in both the nucleus and the cytoplasm.…”

“…Unlike HMG-14 and -17, a substantial portion of HMG-1 and -2, which have been reported to bind preferentially to single-stranded DNA (13)(14)(15)(16); for reviews see 11 and 12), are rather loosely bound to chromatin. They can be solubilized during cell fractionation (17), in particular after repeated exposure of isolated nuclei to 0.14 M NaC1 (18), and in some cells they seem to occur, as suggested from immunofluorescence microscopy, in both compartments, nucleus and cytoplasm (19,20). Whether certain subsets of the HMG proteins 1 and 2, and the homologous protein HMG-T of trout testis, are specifically associated with replicating and/or transcribable regions ofchromatin is so far not clear.…”