Characterization of the distribution and dynamics of chromatin states in the C. elegans germline reveals substantial H3K4me3 remodeling during oogenesis.

Chromatin organization in the C. elegans germline is tightly regulated and critical for germ cell differentiation. Although certain germline epigenetic regulatory mechanisms have been identified, how they influence chromatin structure and ultimately gene expression remains unclear, in part because most genomic studies have focused on data collected from intact worms comprising both somatic and germline tissues. We therefore analyzed histone modification and chromatin accessibility data from isolated germ nuclei representing undifferentiated proliferating and meiosis I populations to define chromatin states. We correlated these states with overall transcript abundance, spatiotemporal expression patterns, and the function of small RNA pathways. Because the essential role of the germline is to transmit genetic information and establish gene expression in the early embryo, we compared epigenetic and transcriptomic profiles from undifferentiated germ cells to those of embryos to define the epigenetic changes during this developmental transition. The active histone modification H3K4me3 shows particularly dynamic remodeling as germ cells differentiate into oocytes, which suggests a mechanism for establishing early transcription of essential genes during zygotic genome activation. This analysis highlights the dynamism of the chromatin landscape across developmental transitions and provides a resource for future investigation into epigenetic regulatory mechanisms in germ cells.

The art of observation: bridging science and art to see the unexpected.

Observation is the heart of research, but it can be challenging to observe deeply and go beyond expected observations. Here, we describe activities designed for scientists to enhance their observational skills by engaging with art. In collaboration with an art gallery at our university, our lab practiced observing representational paintings in a systematic way, separating the act of observation from interpretation. Applying this skill to our microscopy images allowed us to access information in the data that may otherwise have been overlooked. In addition, these activities highlighted the power of collecting observations from multiple observers before generating interpretations, as well as the value of discussing the creative and emotional aspects of data collection and interpretation. We provide concrete examples of how we will incorporate these skills into our research processes, as well as details that other groups can use to engage in similar art-based training activities to enhance their own observational skills.

The Upstream Sequence Transcription Complex dictates nucleosome positioning and promoter accessibility at piRNA genes in the C. elegans germ line.

The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility. In C. elegans and other nematodes, Type-I piRNAs are expressed from >10,000 independently transcribed genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. Clustering of piRNA genes contributes to their germline-specific expression, but the underlying mechanisms are unclear. We analyze isolated germ nuclei to demonstrate that the piRNA genomic domains are located in a heterochromatin-like environment. USTC (Upstream Sequence Transcription Complex) promotes strong association of nucleosomes throughout piRNA clusters, yet organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Localization of USTC to the piRNA domains depends upon the ATPase chromatin remodeler ISW-1, which maintains high nucleosome density across piRNA clusters and ongoing production of piRNA precursors. Overall, this work provides insight into how chromatin states coordinate transcriptional regulation over large genomic domains, with implications for global genome organization.

Nop9 recognizes structured and single-stranded RNA elements of preribosomal RNA.

Nop9 is an essential factor in the processing of preribosomal RNA. Its absence in yeast is lethal, and defects in the human ortholog are associated with breast cancer, autoimmunity, and learning/language impairment. PUF family RNA-binding proteins are best known for sequence-specific RNA recognition, and most contain eight α-helical repeats that bind to the RNA bases of single-stranded RNA. Nop9 is an unusual member of this family in that it contains eleven repeats and recognizes both RNA structure and sequence. Here we report a crystal structure of Saccharomyces cerevisiae Nop9 in complex with its target RNA within the 20S preribosomal RNA. This structure reveals that Nop9 brings together a carboxy-terminal module recognizing the 5' single-stranded region of the RNA and a bifunctional amino-terminal module recognizing the central double-stranded stem region. We further show that the 3' single-stranded region of the 20S target RNA adds sequence-independent binding energy to the RNA-Nop9 interaction. Both the amino- and carboxy-terminal modules retain the characteristic sequence-specific recognition of PUF proteins, but the amino-terminal module has also evolved a distinct interface, which allows Nop9 to recognize either single-stranded RNA sequences or RNAs with a combination of single-stranded and structured elements.

Structural analysis reveals the flexible C-terminus of Nop15 undergoes rearrangement to recognize a pre-ribosomal RNA folding intermediate.

The RNA recognition motif (RRM) is the most abundant RNA-binding domain in eukaryotes, and it plays versatile roles in RNA metabolism. Despite its abundance, diversity of RRM structure and function is generated by variations on a conserved core. Yeast Nop15 is an RRM protein that is essential for large ribosomal subunit biogenesis. We determined a 2.0 Å crystal structure of Nop15 that reveals a C-terminal α-helical region obscures its canonical RNA-binding surface. Small-angle X-ray scattering, NMR and RNA-binding analyses further reveal that the C-terminal residues of Nop15 are highly flexible, but essential for tight RNA binding. Moreover, comparison with a recently reported cryo-electron microscopy structure indicates that dramatic rearrangement of the C-terminal region of Nop15 in the pre-ribosome exposes the RNA-binding surface to recognize the base of its stem-loop target RNA and extends a newly-formed α helix to the distal loop where it forms protein interactions.

Transcriptome analysis uncovers Arabidopsis F-BOX STRESS INDUCED 1 as a regulator of jasmonic acid and abscisic acid stress gene expression.

BACKGROUND: The ubiquitin 26S proteasome system (UPS) selectively degrades cellular proteins, which results in physiological changes to eukaryotic cells. F-box proteins are substrate adaptors within the UPS and are responsible for the diversity of potential protein targets. Plant genomes are enriched in F-box genes, but the vast majority of these have unknown roles. This work investigated the Arabidopsis F-box gene F-BOX STRESS INDUCED 1 (FBS1) for its effects on gene expression in order elucidate its previously unknown biological function. RESULTS: Using publically available Affymetrix ATH1 microarray data, we show that FBS1 is significantly co-expressed in abiotic stresses with other well-characterized stress response genes, including important stress-related transcriptional regulators. This gene suite is most highly expressed in roots under cold and salt stresses. Transcriptome analysis of fbs1-1 knock-out plants grown at a chilling temperature shows that hundreds of genes require FBS1 for appropriate expression, and that these genes are enriched in those having roles in both abiotic and biotic stress responses. Based on both this genome-wide expression data set and quantitative real-time PCR (qPCR) analysis, it is apparent that FBS1 is required for elevated expression of many jasmonic acid (JA) genes that have established roles in combatting environmental stresses, and that it also controls a subset of JA biosynthesis genes. FBS1 also significantly impacts abscisic acid (ABA) regulated genes, but this interaction is more complex, as FBS1 has both positive and negative effects on ABA-inducible and ABA-repressible gene modules. One noteworthy effect of FBS1 on ABA-related stress processes, however, is the restraint it imposes on the expression of multiple class I LIPID TRANSFER PROTEIN (LTP) gene family members that have demonstrated protective effects in water deficit-related stresses. CONCLUSION: FBS1 impacts plant stress responses by regulating hundreds of genes that respond to the plant stress hormones JA and ABA. The positive effect that FBS1 has on JA processes and the negative effect it has on at least some ABA processes indicates that it in part regulates cellular responses balanced between these two important stress hormones. More broadly then, FBS1 may aid plant cells in switching between certain biotic (JA) and abiotic (ABA) stress responses. Finally, because FBS1 regulates a subset of JA biosynthesis and response genes, we conclude that it might have a role in tuning hormone responses to particular circumstances at the transcriptional level.

Oncogenic Kras induces spatiotemporally specific tissue deformation through converting pulsatile into sustained ERK activation.

Tissue regeneration and maintenance rely on coordinated stem cell behaviours. This orchestration can be impaired by oncogenic mutations leading to cancer. However, it is largely unclear how oncogenes perturb stem cells' orchestration to disrupt tissue. Here we used intravital imaging to investigate the mechanisms by which oncogenic Kras mutation causes tissue disruption in the hair follicle. Through longitudinally tracking hair follicles in live mice, we found that KrasG12D, a mutation that can lead to squamous cell carcinoma, induces epithelial tissue deformation in a spatiotemporally specific manner, linked with abnormal cell division and migration. Using a reporter mouse capture real-time ERK signal dynamics at the single-cell level, we discovered that KrasG12D, but not a closely related mutation HrasG12V, converts ERK signal in stem cells from pulsatile to sustained. Finally, we demonstrated that interrupting sustained ERK signal reverts KrasG12D-induced tissue deformation through modulating specific features of cell migration and division.

The Upstream Sequence Transcription Complex Dictates Nucleosome Positioning and Promoter Accessibility at piRNA Genes in the C. elegans Germ Line.

The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility. In C. elegans and other nematodes, Type-I piRNA precursor transcripts are expressed from over 10,000 small, independently regulated genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. These large clusters likely play a significant role in promoting germline-specific expression of piRNAs, but the underlying mechanisms are unclear. By examining the chromatin environment specifically in isolated germ nuclei, we demonstrate that piRNA clusters are located in closed chromatin, and confirm the enrichment for the inactive histone modification H3K27me3. We further show that the piRNA biogenesis factor USTC (Upstream Sequence Transcription Complex) plays two roles - it promotes a strong association of nucleosomes throughout the piRNA clusters, and it organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Overall, this work reveals new insight into how chromatin state coordinates transcriptional regulation over large genomic domains, which has implications for understanding global genome organization in the germ line.

Organ function is preserved despite reorganization of niche architecture in the hair follicle.

The ability of stem cells to build and replenish tissues depends on support from their niche. Although niche architecture varies across organs, its functional importance is unclear. During hair follicle growth, multipotent epithelial progenitors build hair via crosstalk with their remodeling fibroblast niche, the dermal papilla, providing a powerful model to functionally interrogate niche architecture. Through mouse intravital imaging, we show that dermal papilla fibroblasts remodel individually and collectively to form a morphologically polarized, structurally robust niche. Asymmetric TGF-ß signaling precedes morphological niche polarity, and loss of TGF-ß signaling in dermal papilla fibroblasts leads them to progressively lose their stereotypic architecture, instead surrounding the epithelium. The reorganized niche induces the redistribution of multipotent progenitors but nevertheless supports their proliferation and differentiation. However, the differentiated lineages and hairs produced by progenitors are shorter. Overall, our results reveal that niche architecture optimizes organ efficiency but is not absolutely essential for organ function.

Live imaging reveals chromatin compaction transitions and dynamic transcriptional bursting during stem cell differentiation in vivo.

Stem cell differentiation requires dramatic changes in gene expression and global remodeling of chromatin architecture. How and when chromatin remodels relative to the transcriptional, behavioral, and morphological changes during differentiation remain unclear, particularly in an intact tissue context. Here, we develop a quantitative pipeline which leverages fluorescently-tagged histones and longitudinal imaging to track large-scale chromatin compaction changes within individual cells in a live mouse. Applying this pipeline to epidermal stem cells, we reveal that cell-to-cell chromatin compaction heterogeneity within the stem cell compartment emerges independent of cell cycle status, and instead is reflective of differentiation status. Chromatin compaction state gradually transitions over days as differentiating cells exit the stem cell compartment. Moreover, establishing live imaging of Keratin-10 (K10) nascent RNA, which marks the onset of stem cell differentiation, we find that Keratin-10 transcription is highly dynamic and largely precedes the global chromatin compaction changes associated with differentiation. Together, these analyses reveal that stem cell differentiation involves dynamic transcriptional states and gradual chromatin rearrangement.

Maternal Piwi regulates primordial germ cell development to ensure the fertility of female progeny in Drosophila.

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.

Nop9 is a PUF-like protein that prevents premature cleavage to correctly process pre-18S rRNA.

Numerous factors direct eukaryotic ribosome biogenesis, and defects in a single ribosome assembly factor may be lethal or produce tissue-specific human ribosomopathies. Pre-ribosomal RNAs (pre-rRNAs) must be processed stepwise and at the correct subcellular locations to produce the mature rRNAs. Nop9 is a conserved small ribosomal subunit biogenesis factor, essential in yeast. Here we report a 2.1-Å crystal structure of Nop9 and a small-angle X-ray-scattering model of a Nop9:RNA complex that reveals a 'C'-shaped fold formed from 11 Pumilio repeats. We show that Nop9 recognizes sequence and structural features of the 20S pre-rRNA near the cleavage site of the nuclease, Nob1. We further demonstrate that Nop9 inhibits Nob1 cleavage, the final processing step to produce mature small ribosomal subunit 18S rRNA. Together, our results suggest that Nop9 is critical for timely cleavage of the 20S pre-rRNA. Moreover, the Nop9 structure exemplifies a new class of Pumilio repeat proteins.