Cohesins are evolutionarily conserved essential multi-protein complexes that are important for higher-order chromatin organization. They play pivotal roles in the maintenance of genome integrity through mitotic chromosome regulation, DNA repair and replication, as well as gene regulation critical for proper development and cellular differentiation. In this review, we will discuss the multifaceted functions of mammalian cohesins and their apparent functional hierarchy in the cell, with particular focus on their actions in gene regulation and their relevance to human developmental disorders.

We have identified and characterized an alternative RFC complex RFC(Ctf18p, Ctf8p, Dcc1p) that is required for sister chromatid cohesion and faithful chromosome transmission. Ctf18p, Ctf8p, and Dcc1p interact physically in a complex with Rfc2p, Rfc3p, Rfc4p, and Rfc5p but not with Rfc1p or Rad24p. Deletion of CTF18, CTF8, or DCC1 singly or in combination (ctf18Deltactf8Deltadcc1Delta) leads to sensitivity to microtubule depolymerizing drugs and a severe sister chromatid cohesion defect. Furthermore, temperature-sensitive mutations in RFC4 result in precocious sister chromatid separation. Our results highlight a novel function of the RFC proteins and support a model in which sister chromatid cohesion is established at the replication fork via a polymerase switching mechanism and a replication-coupled remodeling of chromatin.

Two chromatin nonhistone proteins (from calf thymus) of the high mobility group, HMG1 and HMG2, reduce the linking number (topological winding number) of a circular DNA if the covalent closure of the DNA is carried out in their presence. This indicates that these proteins can either unwind the double helix, or induce a supercoiling of the DNA.

Hemin has been shown to inhibit specifically the energy-dependent degradation of normal and abnormal proteins in reticulocytes [Etlinger, J. D.& Goldberg, A. L.(1980) J. Biol. Chem. 255, 4563-4568]. The present work demonstrates that the action of hemin involves the multi-enzyme ATP-dependent ubiquitin-dependent proteolytic system exclusively. At a concentration of approximately 25 microM, hemin produces 50% inhibition of the degradation of 125I-labeled bovine serum albumin by this pathway. Hemin has no effect on the basal rate of proteolysis in the absence of either ATP or ubiquitin. At a concentration of hemin that gives complete inhibition of proteolysis, ATP-dependent formation of ubiquitin conjugates continues at about 50% of the control rate but the degradation of these ubiquitin conjugates is completely blocked. Inhibition of overall proteolysis and conjugate degradation are sensitive to hemin concentration to exactly the same extent. Hemin inhibition of conjugate breakdown results in the accumulation of higher molecular weight conjugates that are lost when hemin is removed by dilution. A model is proposed in which hemin acts as a negative allosteric effector in the initial step of a sequential degradative path by which intact ubiquitin conjugates are first cleaved to ubiquitin-associated fragments.

The heavy metal mercury elicits a genetically restricted, anti-nucleolar autoantibody response that targets fibrillarin, a 34-kDa protein component of many small nucleolar ribonucleoprotein particles. The mechanisms by which a toxin such as mercury elicits an autoantibody response that predominantly targets a single intracellular protein autoantigen remain uncertain, but may be prefaced by mercury gaining access to the intracellular environment. Mercury-induced cell death was associated with loss of fibrillarin antigenicity and modification of the molecular properties of fibrillarin as revealed by aberrant migration under nonreducing conditions in SDS-PAGE. Addition of mercury to isolated nuclei also resulted in aberrant migration of fibrillarin, but not other nuclear autoantigens. The sensitivity of the HgCl2-induced modification of fibrillarin to 2-ME, iodoacetamide, and hydrogen peroxide suggested interaction of mercury with the two cysteines in the fibrillarin sequence. This was confirmed by mutation of the cysteines to alanines, which abolished the aberrant migration of fibrillarin in the presence of HgCl2. The modification of the molecular structure of fibrillarin by mercury reduced immunoprecipitation by anti-fibrillarin autoantibodies, pointing to unmodified fibrillarin as the B cell Ag and implicating mercury-modified fibrillarin as the source of T cell antigenicity. These observations demonstrate for the first time that an environmental toxin can alter the physicochemical properties of an autoantigen and may help to explain the antigenic specificity of mercury-induced murine autoimmunity.

In order to study mechanisms and regulation of RNA polymerase II (RNAPII) ubiquitylation and degradation, highly purified factors were used to reconstitute RNAPII ubiquitylation in vitro. We show that arrested RNAPII elongation complexes are the preferred substrates for ubiquitylation. Accordingly, not only DNA-damage-dependent but also DNA-damage-independent transcriptional arrest results in RNAPII ubiquitylation in vivo. Def1, known to be required for damage-induced degradation of RNAPII, stimulates ubiquitylation of RNAPII only in an elongation complex. Ubiquitylation of RNAPII is dependent on its C-terminal repeat domain (CTD). Moreover, CTD phosphorylation at serine 5, a hallmark of the initiating polymerase, but not at serine 2, a hallmark of the elongating polymerase, completely inhibits ubiquitylation. In agreement with this, ubiquitylated RNAPII is hypophosphorylated at serine 5 in vivo, and mutation of the serine 5 phosphatase SSU72 inhibits RNAPII degradation. These results identify several mechanisms that confine ubiquitylation of RNAPII to the forms of the enzyme that arrest during elongation.

We have purified to homogeneity a Drosophila protein which is able to decondense Xenopus sperm chromatin. This protein, which we have called DF 31, is a heat-stable phosphoprotein which displays a molecular weight of 31 kDa on SDS-PAGE, but which has an apparent molecular weight of > 200 kDa on gel filtration. We show that DF 31 decondenses sperm DNA by displacement of sperm-specific proteins. In addition to its sperm decondensation activity, DF 31 is also able to facilitate nucleosome loading on both decondensed sperm DNA and on naked DNA template. The reaction as catalysed by DF 31 is quite efficient; however, the nucleosomes appear to be loaded randomly onto the DNA, not in regular arrays. Although the mechanism by which DF 31 aids nucleosome loading is not yet clear, it most probably occurs through binding of DF 31 to core histones.

Digestion of isolated Friend erythroleukemic cell nuclei with DNase I under conditions which selectively destroy the DNA of transcriptionally "active" genes releases into the supernatant fraction proteins of the non-histone "High Mobility Group"(HMGs). Two of these, HMG-14 and HMG-17(identified by solubility in trichloroacetic acid, electrophoretic mobility on SDS-polyacrylamide gels and by amino acid composition) will partially inhibit the endogenous mouse cell histone deacetylase enzymes when added to in vitro assay mixtures. Other closely related proteins do not share this inhibitory ability and thus the reaction with the enzymes appears to be specific. Since these two HMG proteins appear to be preferentially associated with the "active" fraction of chromatin, these findings have important implications for possible models of eukaryotic gene regulatory mechanisms.

The nonhistone chromatin protein, C-14, was extracted from chromatin of Novikoff hepatoma ascites cells and isolated in high purity as shown by its migration as a single dense spot on two-dimensional polyacrylamide gels. Its mobility on sodium dodecyl sulfate gels is consistent with a molecular weight of approximately 70 000. The amino acid composition shows that protein C-14 has an acidic:basic amino acid ratio of 1.8. Its amino terminal amino acid is lysine. Protein C-14 stimulated the incorporation of [3H]UMP into RNA by approximately 30% when added to naked DNA and homologous RNA polymerase I. A 30% stimulation of [3H]UMP incorporation into RNA was also found when protein C-14 was added to an E. coli RNA polymerase system containing either E. coli or Novikoff hepatoma DNA.

The partial N-terminal amino acid sequence of the antimicrobial peptide reported in the present paper has been submitted to the TrEMBL database under the accession number P83338. A 6.7 kDa antimicrobial peptide was isolated from trout skin secretions using acid extraction followed by cation-exchange chromatography,(t)C(18) solid-phase extraction, and C(18) reversed-phase HPLC. The molecular mass of this peptide, which is tentatively named oncorhyncin III, is 6671 Da, as determined by matrix-assisted laser-desorption ionization MS. N-terminal amino acid sequencing revealed that the first 13 residues of oncorhyncin III are identical with those of the non-histone chromosomal protein H6 from rainbow trout. Hence these data combined with the MS results indicate that oncorhyncin III is likely to be a cleavage product of the non-histone chromosomal protein H6 (residues 1-66) and that it probably contains two methylated residues or one double methylation. The purified peptide exhibits potent antibacterial activity against both Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations in the submicromolar range. The peptide is sensitive to NaCl, and displays no haemolytic activity towards trout erythrocytes at concentrations below 1 microM. Scanning electron microscopy revealed that oncorhyncin III does not cause direct disruption of bacterial cells. Reconstitution of the peptide in planar lipid bilayers strongly disturbs the membranes, but does not induce the formation of stable ion channels. Taken together, these results support the hypothesis that oncorhyncin III plays a role in mucosal innate host defence.

Extracts from mitotic HeLa cells, when injected into Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) as evidenced by the breakdown of the germinal vesicle and the condensation of chromosomes. In this study we have attempted to purify and characterize these mitotic factors. When 0.2 M NaCl-soluble extracts of mitotic HeLa cells were concentrated by ultrafiltration and subjected to affinity chromatography on hydroxylapatite followed by DNA-cellulose, the proteins with MPA eluted as a single peak and their specific activity was increased approx. 200-fold compared with crude extracts. The molecular weight of the mitotic factors was estimated to be 100 kD as determined by chromatography on Sephacryl S-200. SDS-PAGE of the partially-purified mitotic factors indicated the presence of several polypeptides ranging from 40-150 kD with a major band of about 50 kD. The majority of these polypeptides were found to be phosphoproteins as revealed by 32P-labeling and autoradiography. Very little or no phosphorylation was observed at the 50 kD band. Several of these polypeptides were reactive with mitosis-specific monoclonal antibodies, MPM-1 or MPM-2, as shown by immunoblots of these proteins but the major polypeptide band at 50 kD was not. Removal of the immunoreactive polypeptides by precipitation with these antibodies did not destroy the MPA. The MPA of the crude or the partially-purified mitotic factors was destroyed by injection of (but not pretreatment with) alkaline phosphatase within 45 min after injection of mitotic factors. These results are discussed in terms of a possible role of phosphorylation-dephosphorylation of non-histone proteins in the regulation of mitosis and meiosis.