Chromatin analysis of occluded genes.

We recently described two opposing states of transcriptional competency. One is termed 'competent' whereby a gene is capable of responding to trans-acting transcription factors of the cell, such that it is active if appropriate transcriptional activators are present, though it can also be silent if activators are absent or repressors are present. The other is termed 'occluded' whereby a gene is silenced by cis-acting, chromatin-based mechanisms in a manner that blocks it from responding to trans-acting factors, such that it is silent even when activators are present in the cellular milieu. We proposed that gene occlusion is a mechanism by which differentiated cells stably maintain their phenotypic identities. Here, we describe chromatin analysis of occluded genes. We found that DNA methylation plays a causal role in maintaining occlusion for a subset of occluded genes. We further examined a variety of other chromatin marks typically associated with transcriptional silencing, including histone variants, covalent histone modifications and chromatin-associated proteins. Surprisingly, we found that although many of these marks are robustly linked to silent genes (which include both occluded genes and genes that are competent but silent), none is linked specifically to occluded genes. Although the observation does not rule out a possible causal role of these chromatin marks in occlusion, it does suggest that these marks might be secondary effect rather than primary cause of the silent state in many genes.

Systematic identification of cis-silenced genes by trans complementation.

A gene's transcriptional output is the combined product of two inputs: diffusible factors in the cellular milieu acting in trans, and chromatin state acting in cis. Here, we describe a strategy for dissecting the relative contribution of cis versus trans mechanisms to gene regulation. Referred to as trans complementation, it entails fusing two disparate cell types and searching for genes differentially expressed between the two genomes of fused cells. Any differential expression can be causally attributed to cis mechanisms because the two genomes of fused cells share a single homogenized milieu in trans. This assay uncovered a state of transcriptional competency that we termed 'occluded' whereby affected genes are silenced by cis-acting mechanisms in a manner that blocks them from responding to the trans-acting milieu of the cell. Importantly, occluded genes in a given cell type tend to include master triggers of alternative cell fates. Furthermore, the occluded state is maintained during cell division and is extraordinarily stable under a wide range of physiological conditions. These results support the model that the occlusion of lineage-inappropriate genes is a key mechanism of cell fate restriction. The identification of occluded genes by our assay provides a hitherto unavailable functional readout of chromatin state that is distinct from and complementary to gene expression status.

Gliotypic neural stem cells transiently adopt tumorigenic properties during normal differentiation.

An increasing body of evidence suggests that astrocytic gliomas of the central nervous system may be derived from gliotypic neural stem cells. To date, the study of these tumors, particularly the identification of originating cellular population(s), has been frustrated by technical difficulties in accessing the native niche of stem cells. To identify any hallmark signs of cancer in neural stem cells or their progeny, we cultured subventricular zone-derived tissue in a unique in vitro model that temporally and phenotypically recapitulates adult neurogenesis. Contrary to some reports, we found undifferentiated neural stem cells possess few characteristics, suggesting prototumorigenic potential. However, when induced to differentiate, neural stem cells give rise to intermediate progenitors that transiently exhibit multiple glioma characteristics, including aneuploidy, loss of growth-contact inhibition, alterations in cell cycle, and growth factor insensitivity. Further examination of progenitor populations revealed a subset of cells defined by the aberrant expression of (the pathological glioma marker) class III beta-tubulin that exhibit intrinsic parental properties of gliomas, including multilineage differentiation and continued proliferation in the absence of a complex cellular regulatory environment. As tumorigenic characteristics in progenitor cells normally disappear with the generation of mature progeny, this suggests that developmentally intermediate progenitor cells, rather than neural stem cells, may be the origin of so-called "stem cell-derived" tumors.

Adaptability of myosin V studied by simultaneous detection of position and orientation.

We studied the structural dynamics of chicken myosin V by combining the localization power of fluorescent imaging with one nanometer accuracy (FIONA) with the ability to detect angular changes of a fluorescent probe. The myosin V was labeled with bifunctional rhodamine on one of its calmodulin light chains. For every 74 nm translocation, the probe exhibited two reorientational motions, associated with alternating smaller and larger translational steps. Molecules previously identified as stepping alternatively 74-0 nm were found to actually step 64-10 nm. Additional tilting often occurred without full steps, possibly indicating flexibility of the attached myosin heads or probing of their vicinity. Processive myosin V molecules sometimes shifted from the top to the side of actin, possibly to avoid an obstacle. The data indicate marked adaptability of this molecular motor to a nonuniform local environment and provide strong support for a straight-neck model of myosin V in which the lever arm of the leading head is tilted backwards at the prepowerstoke angle.

Nanometer localization of single green fluorescent proteins: evidence that myosin V walks hand-over-hand via telemark configuration.

Myosin V is a homodimeric motor protein involved in trafficking of vesicles in the cell. It walks bipedally along actin filaments, moving cargo approximately 37 nm per step. We have measured the step size of individual myosin heads by fusing an enhanced green fluorescent protein (eGFP) to the N-terminus of one head of the myosin dimer and following the motion with nanometer precision and subsecond resolution. We find the average step size to be 74.1 nm with 9.4 nm (SD) and 0.3 nm (SE). Our measurements demonstrate nanometer localization of single eGFPs, confirm the hand-over-hand model of myosin V procession, and when combined with previous data, suggest that there is a kink in the leading lever arm in the waiting state of myosin V. This kink, or "telemark skier" configuration, may cause strain, which, when released, leads to the powerstroke of myosin, throwing the rear head forward and leading to unidirectional motion.