Probing The Function Of Long Noncoding RNAs In The Nucleus

The nucleus is a highly organized and dynamic environment where regulation and coordination of processes such as gene expression and DNA replication are paramount. In recent years, noncoding RNAs have emerged as key participants in the regulation of nuclear processes. There are a multitude of functional roles for long noncoding RNA (lncRNA), mediated through their ability to act as molecular scaffolds bridging interactions with proteins, chromatin, and other RNA molecules within the nuclear environment. In this review, we discuss the diversity of techniques that have been developed to probe the function of nuclear lncRNAs, along with the ways in which those techniques have revealed insights into their mechanisms of action. Foundational observations into lncRNA function have been gleaned from molecular cytology-based, single-cell approaches to illuminate both the localization and abundance of lncRNAs in addition to their potential binding partners. Biochemical, extraction-based approaches have revealed the molecular contacts between lncRNAs and other molecules within the nuclear environment and how those interactions may contribute to nuclear organization and regulation. Using examples of well-studied nuclear lncRNAs, we demonstrate that the emerging functions of individual lncRNAs have been most clearly deduced from combined cytology and biochemical approaches tailored to study specific lncRNAs. As more functional nuclear lncRNAs continue to emerge, the development of additional technologies to study their interactions and mechanisms of action promise to continually expand our understanding of nuclear organization, chromosome architecture, genome regulation, and disease states

Early Chromosome Condensation By XIST Builds A-Repeat RNA Density That Facilitates Gene Silencing

XIST RNA triggers chromosome-wide gene silencing and condenses an active chromosome into a Barr body. Here, we use inducible human XIST to examine early steps in the process, showing that XIST modifies cytoarchitecture before widespread gene silencing. In just 2–4 h, barely visible transcripts populate the large “sparse zone” surrounding the smaller “dense zone”; importantly, density zones exhibit different chromatin impacts. Sparse transcripts immediately trigger immunofluorescence for H2AK119ub and CIZ1, a matrix protein. H3K27me3 appears hours later in the dense zone, which enlarges with chromosome condensation. Genes examined are silenced after compaction of the RNA/DNA territory. Insights into this come from the findings that the A-repeat alone can silence genes and rapidly, but only where dense RNA supports sustained histone deacetylation. We propose that sparse XIST RNA quickly impacts architectural elements to condense the largely non-coding chromosome, coalescing RNA density that facilitates an unstable, A-repeat-dependent step required for gene silencing

Distinct retroelement classes define evolutionary breakpoints demarcating sites of evolutionary novelty

<p>Abstract</p> <p>Background</p> <p>Large-scale genome rearrangements brought about by chromosome breaks underlie numerous inherited diseases, initiate or promote many cancers and are also associated with karyotype diversification during species evolution. Recent research has shown that these breakpoints are nonrandomly distributed throughout the mammalian genome and many, termed "evolutionary breakpoints" (EB), are specific genomic locations that are "reused" during karyotypic evolution. When the phylogenetic trajectory of orthologous chromosome segments is considered, many of these EB are coincident with ancient centromere activity as well as new centromere formation. While EB have been characterized as repeat-rich regions, it has not been determined whether specific sequences have been retained during evolution that would indicate previous centromere activity or a propensity for new centromere formation. Likewise, the conservation of specific sequence motifs or classes at EBs among divergent mammalian taxa has not been determined.</p> <p>Results</p> <p>To define conserved sequence features of EBs associated with centromere evolution, we performed comparative sequence analysis of more than 4.8 Mb within the tammar wallaby, <it>Macropus eugenii</it>, derived from centromeric regions (CEN), euchromatic regions (EU), and an evolutionary breakpoint (EB) that has undergone convergent breakpoint reuse and past centromere activity in marsupials. We found a dramatic enrichment for long interspersed nucleotide elements (LINE1s) and endogenous retroviruses (ERVs) and a depletion of short interspersed nucleotide elements (SINEs) shared between CEN and EBs. We analyzed the orthologous human EB (14q32.33), known to be associated with translocations in many cancers including multiple myelomas and plasma cell leukemias, and found a conserved distribution of similar repetitive elements.</p> <p>Conclusion</p> <p>Our data indicate that EBs tracked within the class Mammalia harbor sequence features retained since the divergence of marsupials and eutherians that may have predisposed these genomic regions to large-scale chromosomal instability.</p

NPC1 deficiency impairs cerebellar postnatal development of microglia and climbing fiber refinement in a mouse model of Niemann-Pick disease type C.

Little is known about the effects of NPC1 deficiency in brain development and whether these effects contribute to neurodegeneration in Niemann-Pick disease type C (NPC). Degeneration of cerebellar Purkinje cells occurs at an earlier stage and to a greater extent in NPC; therefore, we analyzed the effect of NPC1 deficiency on microglia and on climbing fiber synaptic refinement during cerebellar postnatal development using th

Illumination Pattern Design With Deep Learning For Single-Shot Fourier Ptychographic Microscopy

Fourier ptychographic microscopy allows for the collection of images with a high space-bandwidth product at the cost of temporal resolution. In Fourier ptychographic microscopy, the light source of a conventional widefield microscope is replaced with a light-emitting diode (LED) matrix, and multiple images are collected with different LED illumination patterns. From these images, a higher-resolution image can be computationally reconstructed without sacrificing field-of-view. We use deep learning to achieve single-shot imaging without sacrificing the space-bandwidth product, reducing the acquisition time in Fourier ptychographic microscopy by a factor of 69. In our deep learning approach, a training dataset of high-resolution images is used to jointly optimize a single LED illumination pattern with the parameters of a reconstruction algorithm. Our work paves the way for high-throughput imaging in biological studies

Translating dosage compensation to trisomy 21

Down syndrome is the leading genetic cause of intellectual disabilities, occurring in 1 out of 700 live births. Given that Down syndrome is caused by an extra copy of chromosome 21 that involves over-expression of 400 genes across a whole chromosome, it precludes any possibility of a genetic therapy. Our lab has long studied the natural dosage compensation mechanism for X chromosome inactivation. To “dosage compensate” X-linked genes between females and males, the X-linked XIST gene produces a large non-coding RNA that silences one of the two X chromosomes in female cells. The initial motivation of this study was to translate the natural mechanisms of X chromosome inactivation into chromosome therapy for Down syndrome. Using genome editing with zinc finger nucleases, we have successfully inserted a large XIST transgene into Chromosome 21 in Down syndrome iPS cells, which results in chromosome-wide transcriptional silencing of the extra Chromosome 21. Remarkably, deficits in proliferation and neural growth are rapidly reversed upon silencing one chromosome 21. Successful trisomy silencing in vitro surmounts the major first step towards potential development of “chromosome therapy” for Down syndrome. The human iPSC-based trisomy correction system we established opens a unique opportunity to identify therapeutic targets and study transplantation therapies for Down syndrome

Ectopic Expression Of Pericentric HSATII RNA Results In Nuclear RNA Accumulation, MeCP2 Recruitment, And Cell Division Defects

Within the pericentric regions of human chromosomes reside large arrays of tandemly repeated satellite sequences. Expression of the human pericentric satellite HSATII is prevented by extensive heterochromatin silencing in normal cells, yet in many cancer cells, HSATII RNA is aberrantly expressed and accumulates in large nuclear foci in cis. Expression and aggregation of HSATII RNA in cancer cells is concomitant with recruitment of key chromatin regulatory proteins including methyl-CpG binding protein 2 (MeCP2). While HSATII expression has been observed in a wide variety of cancer cell lines and tissues, the effect of its expression is unknown. We tested the effect of stable expression of HSATII RNA within cells that do not normally express HSATII. Ectopic HSATII expression in HeLa and primary fibroblast cells leads to focal accumulation of HSATII RNA in cis and triggers the accumulation of MeCP2 onto nuclear HSATII RNA bodies. Further, long-term expression of HSATII RNA leads to cell division defects including lagging chromosomes, chromatin bridges, and other chromatin defects. Thus, expression of HSATII RNA in normal cells phenocopies its nuclear accumulation in cancer cells and allows for the characterization of the cellular events triggered by aberrant expression of pericentric satellite RNA

Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development.

BACKGROUND: We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development. RESULTS: The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements. CONCLUSIONS: Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution

Pericentromeric Satellites: Implications For Chromosome Evolution And Misregulation In Disease

Nearly half of the human genome consists of noncoding repetitive DNA elements, including tandem satellite repeats in large blocks at the pericentromeric regions of chromosomes and intergenic repetitive elements. While both repeat types were long thought to remain mostly silent, recent evidence indicates that repeats can be expressed, but the extent and regulation of their expression or their potential function(s) remain to be elucidated. The location of satellite sequences within pericentromeric regions is largely conserved; this is in contrast to the apparent lack of sequence conservation among even closely related species. Due to their critical location within regions vital for cell division, it is expected that tight regulation of pericentromeric satellite sequences is essential for both epigenetic and genetic stability. We have developed tools to examine the effect of pericentromeric satellite expression on cell division and the distribution of key regulatory proteins. Our data suggests induced expression of pericentromeric satellite RNA is tightly linked to both epigenetic instability and aberrant cell division. Further, using a comparative genomics approach, we examine the evolutionary conservation of a subset of satellite sequences found to be particularly prone to misregulation in cancer

The role of retroviruses and RNA in mammalian centromere competency

The transcriptional framework of the eukaryotic centromere core has been described in budding yeast and rice, but for most eukaryotes and all vertebrates it remains unknown. The lack of large pericentric repeats in the tammar wallaby has made it possible to map the genomic features at the centromere core in a mammalian species for the first time. The centromere of the tammar wallaby is comprised of interspersed arrays of satellite and retroviral DNA. This thesis reports on the finding of a novel size class of small RNA produced from satellite and retroviral transcriptional units at the centromere. Deep sequencing reveals these small RNAs are enriched for retroelements and retroelement-contiguous sequences at the centromere. Results implicate a role for retroviral promoters in transcription of this new class of small RNA and indicate a role in centromere function and regulation of cell division. The discovery of this new RNA form, transcribed from mammalian centromeres, brings together several independent lines of evidence that point to a conserved, retroviral-encoded RNA entity at the core of mammalian centromeres.