The hierarchical packaging of eukaryotic chromatin plays a central role in transcriptional regulation and other DNA-related biological processes. Here, we report the 11-angstrom-resolution cryogenic electron microscopy (cryo-EM) structures of 30-nanometer chromatin fibers reconstituted in the presence of linker histone H1 and with different nucleosome repeat lengths. The structures show a histone H1-dependent left-handed twist of the repeating tetranucleosomal structural units, within which the four nucleosomes zigzag back and forth with a straight linker DNA. The asymmetric binding and the location of histone H1 in chromatin play a role in the formation of the 30-nanometer fiber. Our results provide mechanistic insights into how nucleosomes compact into higher-order chromatin fibers.

The eukaryotic 20S proteasome is responsible for the degradation of many cellular proteins, but how it is assembled and how its distinct active sites are formed are not understood. Like other proteasome subunits, the yeast Doa3 protein is synthesized in precursor form. We show that the N-terminal propeptide is required for Doa3 incorporation into the proteasome and, remarkably, that the propeptide functions in trans, suggesting it serves a chaperone-like function in proteasome biogenesis. Propeptide processing is not required for proteasome assembly but is needed for maturation of a specific subset of active sites. The likely nucleophile for these sites is provided by the N-terminal threonine of mature Doa3. Additional data indicate that precursor processing is autocatalytic and requires association of the two halves of the proteasome particle, thereby preventing formation of proteolytic sites until the central hydrolytic chamber has been sealed off from the rest of the cell.

The mammalian auditory sensory organ, the organ of Corti, consists of sensory hair cells with uniformly oriented stereocilia on the apical surfaces, displaying a distinct planar cell polarity (PCP) parallel to the sensory epithelium 1-3 . It is not clear how this polarity is achieved during differentiation 4-5 . Here we show that the organ of Corti is formed from a thicker and shorter postmitotic primordium through unidirectional extension, characteristic of cellular intercalation known as convergent extension 6 . Mutations in the PCP pathway interfere with this extension, resulting a shortened and widened cochlea, as well as misorientation of stereocilia. Furthermore, parallel to the homologous pathway in Drosophila 7,8 , a mammalian PCP component Dishevelled2 displays PCPdependent polarized subcellular localization across the organ of Corti. Together, these data suggest a conserved molecular mechanism for PCP pathways in invertebrates and vertebrates, and indicate that the mammalian PCP pathway might directly couple cellular intercalations to PCP establishment in the cochlea.Between embryonic day 13 (E13) and E14, precursors for the organ of Corti can be identified as a zone of non-proliferating cells within the cochlear duct using BrdU pulse-labeling (Fig. 1c, brackets) 4,9-10 . Subsequently, a gradient of cell differentiation within the precursor domain initiates near the base of the cochlea and leads to the patterning of one row of inner and three rows of outer hair cells (Fig. 1d , 1e) along the entire length of the cochlea by E18.5 10 , with uniformly oriented stereocilia on the apical surface of hair cells (Fig. 1e, green). From E14.5 to E18.5, the length of the cochlea is increased approximately 2 fold ( Fig. 1a-b). As viewed in cross sections, the developing organ of Corti in the same period is thinned from a 4-5 cell layered primordium (Fig. 1g) to only two layers of cells (Fig. 1h). One layer of hair cells lies above a layer of supporting cell nuclei whose cytoplasmic phalangeal processes reach the apexes of the hair cells, separating them from each other (Fig. 1h). Based on these observations, we have previously proposed that the longer and thinner mature organ is formed from a defined number of postmitotic precursor cells through integrated cellular intercalation movements 4 . BrdU chasing from E14 to E18 further indicated that there was no detectable cell mixing between the primordial organ of Corti and the surrounding cells (Fig. 1d) the hair cells at E14.5 marked by red fluorescent protein (Math1/RFP) were multi-cell layered ( Fig. 1f) within the sensory primordium (Fig. 1g). The thinning of the sensory primordium is accomplished by E18.5 without detectable cell death ( Fig. 1g-h).To further test our hypothesis, we bisected E14.5 cochlear epithelia into apical and basal portions, cultured them in a defined media. Using the cutting wound sites as reference points, we monitored the extension of the organ of Corti from apical-to-basal or basal-to-apical directions for the apical cultures ...

Hearing impairment due to the loss of sensory hair cells is permanent in humans. Considerable interest targets the hair cell differentiation factor Atoh1 as a potential tool with which to promote hair cell regeneration. We generated a novel mouse model to direct the expression of Atoh1 in a spatially and temporally specific manner in the postnatal mammalian cochlea to determine the competency of various types of cochlear epithelial cells for hair cell differentiation. Atoh1 can generate cells in young animals with morphological, molecular, and physiological properties reminiscent of hair cells.This competency is cell type specific and progressively restricted with age. Significantly, Atoh1 induces ectopic sensory patches through Notch signaling to form a cellular mosaic similar to the endogenous sensory epithelia and expansion of the sensory mosaic through the conversion of supporting cells and nonautonomous supporting cell production. Furthermore, Atoh1 also activates proliferation within the normally postmitotic cochlear epithelium. These results provide insight into the potential and limitations of Atoh1-mediated hair cell regeneration.

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. Cochlear gap junctions coassembled from Cx26 and 30 show faster intercellular Ca 2ϩ signaling than homomeric counterparts.

Several basic helix-loop-helix (bHLH) genes have been shown to be essential for the generation of the auditory sensory hair cells or the spiral ganglion (SG) neurons that innervate the hair cells in the cochlea, as well as a variety of cell types in the other nervous systems. However, it remains elusive what cellular context-dependent mechanisms confer the inner ear-specific neuronal or sensory competency/identities. We explored the possibility that one of the mechanisms responsible for generating cellular diversity in the nervous system through cooperative action of bHLH and LIM-homeodomain (LIM-HD) transcriptional factors might also contribute to the inner ear-specific sensory and/or neuronal competency. Here, we show that Islet1 (Isl1), a LIM-HD protein, is expressed early in the otocyst in the region that gives rise to both the auditory sensory organ, the organ of Corti, and SG neurons. Subsequently, the expression of Isl1 is maintained in SG neurons but is transitory in the sensory lineage. At embryonic day 12 (E12) in mice, the expression of Isl1 marks distinctively the ventral portion of the nascent cochlear epithelium encompassing the primordial organ of Corti. At E13, Isl1 is maintained at relatively high levels in the sensory primordium while down-regulated in the other regions of the cochlear duct. As the sensory epithelium starts to differentiate, it is down-regulated in the entire cochlear epithelium. The expression of Isl1 in the developing inner ear reveals an early and likely a common step in the development of both sensory and neuronal lineages of the inner ear, and suggests its potential role in the inner ear-specific sensory and neuronal cell development.

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