Migration cues induce chromatin alterations

Directed cell migration is a property central to multiple basic biological processes. Here, we show that directed cell migration is associated with global changes in the chromatin fiber. Polarized posttranslational changes in histone H1 along with a transient decrease in H1 mobility were detected in cells facing the scratch in a wound healing assay. In parallel to the changes in H1, the levels of the heterochromatin marker histone H3 lysine 9 tri-methylation were elevated. Interestingly, reduction of the chromatin-binding affinity of H1 altered the cell migration rates. Moreover, migration-associated changes in histone H1 were observed during nuclear motility in the simple multicellular organism Neurospora crassa . Our studies suggest that dynamic reorganization of the chromatin fiber is an early event in the cellular response to migration cues.     

Defects in CD8+ regulatory T cells in the lamina propria of patients with inflammatory bowel disease

Mucosal tolerance is believed to be partly mediated by regulatory T cells. Intestinal epithelial cells (IECs) may play an important role in the generation of such regulatory cells, because they are able to process and present Ag to T cells. Furthermore, we have previously demonstrated that IECs are able to generate regulatory CD8(+) T cells in vitro. In the present study, we have analyzed lamina propria (LP) lymphocytes for the presence of such regulatory CD8(+) T cells in normal individuals as well as in patients with inflammatory bowel disease (IBD). The results of the present study show that LP CD8(+) T cells derived from normal controls possess regulatory activity, whereas both unfractionated LP lymphocytes and purified LP CD4(+) T cells do not. The LP CD8(+) T cells suppress Ig production by pokeweed mitogen-stimulated PBMCs by 31-80%, in a cell contact-dependent manner. No significant difference in suppression between CD28(+) and CD28(-)CD8(+) LP T cells was observed. In contrast to CD8(+) T cells from normal LP, CD8(+) T cells isolated from LP of IBD patients, did not suppress Ig production by pokeweed mitogen-stimulated PBMC (five of six ulcerative colitis specimens; six of six Crohn's disease specimens). Furthermore, we demonstrate that the frequency of TCR Vbeta5.1-positive CD8(+) T cells, which we previously have demonstrated to be regulatory and to be expanded by IECs in vitro, is decreased in IBD LP compared with normal LP. In conclusion, this study demonstrates that CD8(+) T cells with regulatory activity are present in the LP of normal healthy individuals, but not in patients with IBD, suggesting that these cells might play an active role in mucosal tolerance.     

Genetic synthetic lethality screen at the single gene level in cultured human cells

Recently, we demonstrated the feasibility of a chemical synthetic lethality screen in cultured human cells. We now demonstrate the principles for a genetic synthetic lethality screen. The technology employs both an immortalized human cell line deficient in the gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest, and the use of a novel GFP-based double-label fluorescence system. Dominant negative genetic suppressor elements (GSEs) are selected from an episomal library expressing short truncated sense and antisense cDNAs for a gene likely to be synthetic lethal with the gene of interest. Expression of these GSEs prevents spontaneous loss of the GFP-marked episomal survival plasmid, thus allowing FACS enrichment for cells retaining the survival plasmid (and the GSEs). The dominant negative nature of the GSEs was validated by the decreased resident enzymatic activity present in cells harboring the GSEs. Also, cells mutated in the gene of interest exhibit reduced survival upon GSE expression. The identification of synthetic lethal genes described here can shed light on functional genetic interactions between genes involved in normal cell metabolism and in disease.     

Characterization of recombinant murine leukemia virus integrase.

Retroviral integration involves two DNA substrates that play different roles. The viral DNA substrate is recognized by virtue of specific nucleotide sequences near the end of a double-stranded DNA molecule. The target DNA substrate is recognized at internal sites with little sequence preference; nucleosomal DNA appears to be preferred for this role. Despite this apparent asymmetry in the sequence, structure, and roles of the DNA substrates in the integration reaction, the existence of distinct binding sites for viral and target DNA substrates has been controversial. In this report, we describe the expression in Escherichia coli and purification of Moloney murine leukemia virus integrase as a fusion protein with glutathione S-transferase, characterization of its activity by using several model DNA substrates, and the initial kinetic characterization of its interactions with a model viral DNA substrate. We provide evidence for functionally and kinetically distinct binding sites for viral and target DNA substrates and describe a cross-linking assay for DNA binding at a site whose specificity is consistent with the target DNA binding site.     

Recruitment of effector lymphocytes by initiator lymphocytes. Circulating lymphocytes are trapped in the reacting lymph node.

In previous studies, we have shown that initiator T lymphocytes (ITL) sensitized in vitro recruit effector T lymphocytes in vivo. When ITL were sensitized against fibroblast antigens in vitro and injected into footpads of syngeneic recipients, they induced enlargement of the draining popliteal lymph node (PLN) and the development there of specific effector lymphocytes of recipient origin. To study the basis of this lymph node response in recruitment, we injected 51Cr-labeled spleen cells i.v. into recipients of sensitized ITL and found that the labeled circulating lymphocytes were trapped in the reacting PLN. The trapping depended on surface properties of the labeled circulating lymphocytes, as revealed by various enzymatic treatments. The trapping process was radiosensitive, both on the part of the trapped lymphocytes and the lymph node-trapping mechanism. Thus, sensitized ITL injected into the hind footpads migrate to the PLN and induce the trapping of circulating recruitable lymphocytes, which either differentiate into or regulate the differentiation of effector T lymphocytes.