MukB-mediated Catenation of DNA Is ATP and MukEF Independent.

Properly condensed chromosomes are necessary for accurate segregation of the sisters after DNA replication. The Escherichia coli condesin is MukB, a structural maintenance of chromosomes (SMC)-like protein, which forms a complex with MukE and the kleisin MukF. MukB is known to be able to mediate knotting of a DNA ring, an intramolecular reaction. In our investigations of how MukB condenses DNA we discovered that it can also mediate catenation of two DNA rings, an intermolecular reaction. This activity of MukB requires DNA binding by the head domains of the protein but does not require either ATP or its partner proteins MukE or MukF. The ability of MukB to mediate DNA catenation underscores its potential for bringing distal regions of a chromosome together.

The MukB-ParC interaction affects the intramolecular, not intermolecular, activities of topoisomerase IV.

Proper chromosome organization is accomplished through binding of proteins such as condensins that shape the DNA and by modulation of chromosome topology by the action of topoisomerases. We found that the interaction between MukB, the bacterial condensin, and ParC, a subunit of topoisomerase IV, enhanced relaxation of negatively supercoiled DNA and knotting by topoisomerase IV, which are intramolecular DNA rearrangements but not decatenation of multiply linked DNA dimers, which is an intermolecular DNA rearrangement required for proper segregation of daughter chromosomes. MukB DNA binding and a specific chiral arrangement of the DNA was required for topoisomerase IV stimulation because relaxation of positively supercoiled DNA was unaffected. This effect could be attributed to a more effective topological reconfiguration of the negatively supercoiled compared with positively supercoiled DNA by MukB. These data suggest that the MukB-ParC interaction may play a role in chromosome organization rather than in separation of daughter chromosomes.

Physical and functional interaction between the condensin MukB and the decatenase topoisomerase IV in Escherichia coli.

Proper geometric and topological organization of DNA is essential for all chromosomal processes. Two classes of proteins play major roles in organizing chromosomes: condensin complexes and type II topoisomerases. In Escherichia coli, MukB, a structural maintenance of chromosome-like component of the bacterial condensin, and topoisomerase IV (Topo IV), a type II topoisomerase that decatenates the newly replicated daughter chromosomes, are both essential for chromosome segregation in rapidly growing cells. However, little is known about the interplay between MukB and Topo IV. Here we demonstrate a physical and functional interaction between MukB and ParC, a subunit of Topo IV, in vitro. The site of MukB interaction was located on the C-terminal domain of ParC and a loss-of-interaction mutant, ParC R705E R729A, was isolated. This variant retained full activity as a topoisomerase when reconstituted with ParE to form Topo IV. We show that MukB stimulates the superhelical DNA relaxation activity of wild-type Topo IV, but not that of Topo IV reconstituted with ParC R705E R729A.

Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation.

Peripheral blood neutrophils form highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs), that have been implicated in innate immune response to bacterial infection. Neutrophils express high levels of peptidylarginine deiminase 4 (PAD4), which catalyzes histone citrullination. However, whether PAD4 or histone citrullination plays a role in chromatin structure in neutrophils is unclear. In this study, we show that the hypercitrullination of histones by PAD4 mediates chromatin decondensation. Histone hypercitrullination is detected on highly decondensed chromatin in HL-60 granulocytes and blood neutrophils. The inhibition of PAD4 decreases histone hypercitrullination and the formation of NET-like structures, whereas PAD4 treatment of HL-60 cells facilitates these processes. The loss of heterochromatin and multilobular nuclear structures is detected in HL-60 granulocytes after PAD4 activation. Importantly, citrullination of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures. Together, these results suggest that histone hypercitrullination has important functions in chromatin decondensation in granulocytes/neutrophils.

Cell lineage-specific interactions between Men1 and Rb in neuroendocrine neoplasia.

Inactivation of multiple endocrine neoplasia (MEN) type 1 gene (Men1) results in development of multiple endocrine tumors in Men1(+/-) mice and in humans. Intriguingly, loss of the wild-type retinoblastoma 1 (Rb) gene also leads to MEN-like phenotype in Rb(+/-) mice. To evaluate potential genetic interactions between these genes, we prepared and characterized Men1(+/-)Rb(+/-) compound mice in parallel with their parental genotypes. Men1 and Rb did not cooperate in tumor suppression, as demonstrated by comparable survival rates of Rb(+/-) and Men1(+/-)Rb(+/-) mice, absence of tumor growth acceleration and lack of novel neoplasms. Notably, the loss of the remaining copy of the wild-type Men1 and Rb was mutually exclusive in all tumors of Men1(+/-)Rb(+/-) mice, including pituitary anterior lobe and adrenal medulla neoplasms shared by Rb- and Men1-deficient phenotypes. Down-regulation of Men1 targets p18 and p27 and increased presence of phosphorylated-Rb were observed in Men1-deficient pheochromocytomas of Men1(+/-)Rb(+/-) and Men1(+/-) mice. At the same time, the RNA interference (RNAi) knock-down of Men1 mRNA resulted in increased apoptosis of Rb-deficient medullary thyroid carcinoma cells. These results demonstrate that, depending on cell lineage context, combined Men1 and Rb deficiency may be either redundant or detrimental to neoplastic growth. Identification of cell lineage-specific interactions between Men1 and Rb may have important implications for development of rationally designed therapeutic approaches.