Mapping pediatric brain tumors to their origins in the developing cerebellum.

BACKGROUND: Distinguishing the cellular origins of childhood brain tumors is key for understanding tumor initiation and identifying lineage-restricted, tumor-specific therapeutic targets. Previous strategies to map the cell-of-origin typically involved comparing human tumors to murine embryonal tissues, which is potentially limited due to species-specific differences. The aim of this study was to unravel the cellular origins of the 3 most common pediatric brain tumors, ependymoma, pilocytic astrocytoma, and medulloblastoma, using a developing human cerebellar atlas. METHODS: We used a single-nucleus atlas of the normal developing human cerebellum consisting of 176 645 cells as a reference for an in-depth comparison to 4416 bulk and single-cell transcriptome tumor datasets, using gene set variation analysis, correlation, and single-cell matching techniques. RESULTS: We find that the astroglial cerebellar lineage is potentially the origin for posterior fossa ependymomas. We propose that infratentorial pilocytic astrocytomas originate from the oligodendrocyte lineage and MHC II genes are specifically enriched in these tumors. We confirm that SHH and Group 3/4 medulloblastomas originate from the granule cell and unipolar brush cell lineages. Radiation-induced gliomas stem from cerebellar glial lineages and demonstrate distinct origins from the primary medulloblastoma. We identify tumor genes that are expressed in the cerebellar lineage of origin, and genes that are tumor specific; both gene sets represent promising therapeutic targets for future study. CONCLUSION: Based on our results, individual cells within a tumor may resemble different cell types along a restricted developmental lineage. Therefore, we suggest that tumors can arise from multiple cellular states along the cerebellar "lineage of origin."

Chemical and Microstructural Characterization of pH and [Ca2+] Dependent Sol-Gel Transitions in Mucin Biopolymer.

Mucus is responsible for controlling transport and barrier function in biological systems, and its properties can be significantly affected by compositional and environmental changes. In this study, the impacts of pH and CaCl2 were examined on the solution-to-gel transition of mucin, the primary structural component of mucus. Microscale structural changes were correlated with macroscale viscoelastic behavior as a function of pH and calcium addition using rheology, dynamic light scattering, zeta potential, surface tension, and FTIR spectroscopic characterization. Mucin solutions transitioned from solution to gel behavior between pH 4-5 and correspondingly displayed a more than ten-fold increase in viscoelastic moduli. Addition of CaCl2 increased the sol-gel transition pH value to ca. 6, with a twofold increase in loss moduli at low frequencies and ten-fold increase in storage modulus. Changing the ionic conditions-specifically [H+] and [Ca2+] -modulated the sol-gel transition pH, isoelectric point, and viscoelastic properties due to reversible conformational changes with mucin forming a network structure via  non-covalent cross-links between mucin chains.

Deletion of DXZ4 on the human inactive X chromosome alters higher-order genome architecture.

During interphase, the inactive X chromosome (Xi) is largely transcriptionally silent and adopts an unusual 3D configuration known as the "Barr body." Despite the importance of X chromosome inactivation, little is known about this 3D conformation. We recently showed that in humans the Xi chromosome exhibits three structural features, two of which are not shared by other chromosomes. First, like the chromosomes of many species, Xi forms compartments. Second, Xi is partitioned into two huge intervals, called "superdomains," such that pairs of loci in the same superdomain tend to colocalize. The boundary between the superdomains lies near DXZ4, a macrosatellite repeat whose Xi allele extensively binds the protein CCCTC-binding factor. Third, Xi exhibits extremely large loops, up to 77 megabases long, called "superloops." DXZ4 lies at the anchor of several superloops. Here, we combine 3D mapping, microscopy, and genome editing to study the structure of Xi, focusing on the role of DXZ4 We show that superloops and superdomains are conserved across eutherian mammals. By analyzing ligation events involving three or more loci, we demonstrate that DXZ4 and other superloop anchors tend to colocate simultaneously. Finally, we show that deleting DXZ4 on Xi leads to the disappearance of superdomains and superloops, changes in compartmentalization patterns, and changes in the distribution of chromatin marks. Thus, DXZ4 is essential for proper Xi packaging.

Two novel DXZ4-associated long noncoding RNAs show developmental changes in expression coincident with heterochromatin formation at the human (Homo sapiens) macrosatellite repeat.

On the male X and female active X chromosome (Xa), the macrosatellite repeat (MSR) DXZ4 is packaged into constitutive heterochromatin characterized by CpG methylation and histone H3 tri-methylated at lysine-9 (H3K9me3). In contrast, DXZ4 on the female inactive X chromosome (Xi), is packaged into euchromatin, is bound by the architectural protein CCCTC-binding factor, and mediates Xi-specific long-range cis contact with similarly packaged tandem repeats on the Xi. In cancer, male DXZ4 can inappropriately revert to a Xi-like state and other MSRs have been reported to adopt alternate chromatin configurations in response to disease. Given this plasticity, we sought to identify factors that might control heterochromatin at DXZ4. In human embryonic stem cells, we found low levels of 5-hydroxymethylcytosine at DXZ4 and that this mark is lost upon differentiation as H3K9me3 is acquired. We identified two previously undescribed DXZ4 associated noncoding transcripts (DANT1 and DANT2) that are transcribed toward DXZ4 from promoters flanking the array. Each generates transcript isoforms that traverse the MSR. However, upon differentiation, enhancer of Zeste-2 silences DANT1, and DANT2 transcription terminates prior to entering DXZ4. These data support a model wherein DANT1 and/or DANT2 may function to regulate constitutive heterochromatin formation at this MSR.

A region of euchromatin coincides with an extensive tandem repeat on the mouse (Mus musculus) inactive X chromosome.

Euchromatic features are largely absent from the human inactive X chromosome (Xi), with the exception of several large tandem repeats that can be detected as euchromatin bands at metaphase. Despite residing megabases apart, these tandem repeats make frequent inactive X-specific interactions. The mouse homologue has been reported for at least one of the tandem repeats, but whether the mouse Xi is also characterized by distinct bands of euchromatin remains unknown. We examined the mouse Xi for the presence of euchromatin bands by examining the pattern of histone H3 dimethylated at lysine 4 and detected two major signals. The first band resides in the subtelomeric region of band XF5 and may correspond to the pseudoautosomal region. The second band localizes to XE3 and coincides with an extensive complex repeat composed of a large tandem and inverted repeat segment as well as several large short interspersed nuclear element (SINE)-rich tandem repeats. Fluorescence in situ hybridization reveals that sequences with homology to the repeat region are scattered along the length of the Y chromosome. Immunofluorescence analysis of histone H3 trimethylated at lysine 9 on metaphase chromosomes indicates that the repeat region corresponds to a band of constitutive heterochromatin on the male X and female active X chromosomes, whereas the euchromatin signal appears to be female specific. These data suggest that the band of euchromatin observed at XE3 is unique to the mouse Xi, comparable to the chromatin arrangement of several large tandem repeats located on the human X chromosome.

A novel tRNA variable number tandem repeat at human chromosome 1q23.3 is implicated as a boundary element based on conservation of a CTCF motif in mouse.

The human genome contains numerous large tandem repeats, many of which remain poorly characterized. Here we report a novel transfer RNA (tRNA) tandem repeat on human chromosome 1q23.3 that shows extensive copy number variation with 9-43 repeat units per allele and displays evidence of meiotic and mitotic instability. Each repeat unit consists of a 7.3 kb GC-rich sequence that binds the insulator protein CTCF and bears the chromatin hallmarks of a bivalent domain in human embryonic stem cells. A tRNA containing tandem repeat composed of at least three 7.6-kb GC-rich repeat units reside within a syntenic region of mouse chromosome 1. However, DNA sequence analysis reveals that, with the exception of the tRNA genes that account for less than 6% of a repeat unit, the remaining 7.2 kb is not conserved with the notable exception of a 24 base pair sequence corresponding to the CTCF binding site, suggesting an important role for this protein at the locus.

The mitochondrial genomes of Amphiascoides atopus and Schizopera knabeni (Harpacticoida: Miraciidae) reveal similarities between the copepod orders Harpacticoida and Poecilostomatoida.

Members of subclass Copepoda are abundant, diverse, and-as a result of their variety of ecological roles in marine and freshwater environments-important, but their phylogenetic interrelationships are unclear. Recent studies of arthropods have used gene arrangements in the mitochondrial (mt) genome to infer phylogenies, but for copepods, only seven complete mt genomes have been published. These data revealed several within-order and few among-order similarities. To increase the data available for comparisons, we sequenced the complete mt genome (13,831base pairs) of Amphiascoides atopus and 10,649base pairs of the mt genome of Schizopera knabeni (both in the family Miraciidae of the order Harpacticoida). Comparison of our data to those for Tigriopus japonicus (family Harpacticidae, order Harpacticoida) revealed similarities in gene arrangement among these three species that were consistent with those found within and among families of other copepod orders. Comparison of the mt genomes of our species with those known from other copepod orders revealed the arrangement of mt genes of our Harpacticoida species to be more similar to that of Sinergasilus polycolpus (order Poecilostomatoida) than to that of T. japonicus. The similarities between S. polycolpus and our species are the first to be noted across the boundaries of copepod orders and support the possibility that mt-gene arrangement might be used to infer copepod phylogenies. We also found that our two species had extremely truncated transfer RNAs and that gene overlaps occurred much more frequently than has been reported for other copepod mt genomes.

Boosting transcription by transcription: enhancer-associated transcripts.

Enhancers are traditionally viewed as DNA sequences located some distance from a promoter that act in cis and in an orientation-independent fashion to increase utilization of specific promoters and thereby regulate gene expression. Much progress has been made over the last decade toward understanding how these distant elements interact with target promoters, but how transcription is enhanced remains an object of active inquiry. Recent reports convey the prevalence and diversity of enhancer transcription and transcripts and support both as key factors with mechanistically distinct, but not mutually exclusive roles in enhancer function. Decoupling the causes and effects of transcription on the local chromatin landscape and understanding the role of enhancer transcripts in the context of long-range interactions are challenges that require additional attention. In this review, we focus on the possible functions of enhancer transcription by highlighting several recent enhancer RNA papers and, within the context of other enhancer studies, speculate on the role of enhancer transcription in regulating differential gene expression.