Genomic data resources of the Brain Somatic Mosaicism Network for neuropsychiatric diseases.

Somatic mosaicism is defined as an occurrence of two or more populations of cells having genomic sequences differing at given loci in an individual who is derived from a single zygote. It is a characteristic of multicellular organisms that plays a crucial role in normal development and disease. To study the nature and extent of somatic mosaicism in autism spectrum disorder, bipolar disorder, focal cortical dysplasia, schizophrenia, and Tourette syndrome, a multi-institutional consortium called the Brain Somatic Mosaicism Network (BSMN) was formed through the National Institute of Mental Health (NIMH). In addition to genomic data of affected and neurotypical brains, the BSMN also developed and validated a best practices somatic single nucleotide variant calling workflow through the analysis of reference brain tissue. These resources, which include >400 terabytes of data from 1087 subjects, are now available to the research community via the NIMH Data Archive (NDA) and are described here.

Comprehensive identification of somatic nucleotide variants in human brain tissue.

BACKGROUND: Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. RESULTS: Here, the Brain Somatic Mosaicism Network conducts a coordinated, multi-institutional study to examine the ability of existing methods to detect simulated somatic single-nucleotide variants (SNVs) in DNA mixing experiments, generate multiple replicates of whole-genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual, devise strategies to discover somatic SNVs, and apply various approaches to validate somatic SNVs. These efforts lead to the identification of 43 bona fide somatic SNVs that range in variant allele fractions from ~ 0.005 to ~ 0.28. Guided by these results, we devise best practices for calling mosaic SNVs from 250× whole-genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrate that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. CONCLUSIONS: This study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

EKLF/KLF1 expression defines a unique macrophage subset during mouse erythropoiesis.

Erythroblastic islands are a specialized niche that contain a central macrophage surrounded by erythroid cells at various stages of maturation. However, identifying the precise genetic and transcriptional control mechanisms in the island macrophage remains difficult due to macrophage heterogeneity. Using unbiased global sequencing and directed genetic approaches focused on early mammalian development, we find that fetal liver macrophages exhibit a unique expression signature that differentiates them from erythroid and adult macrophage cells. The importance of erythroid Krüppel-like factor (EKLF)/KLF1 in this identity is shown by expression analyses in EKLF-/- and in EKLF-marked macrophage cells. Single-cell sequence analysis simplifies heterogeneity and identifies clusters of genes important for EKLF-dependent macrophage function and novel cell surface biomarkers. Remarkably, this singular set of macrophage island cells appears transiently during embryogenesis. Together, these studies provide a detailed perspective on the importance of EKLF in the establishment of the dynamic gene expression network within erythroblastic islands in the developing embryo and provide the means for their efficient isolation.

Cocaine-Induced Chromatin Modifications Associate With Increased Expression and Three-Dimensional Looping of Auts2.

BACKGROUND: Exposure to drugs of abuse alters the epigenetic landscape of the brain's reward regions, such as the nucleus accumbens. We investigated how combinations of chromatin modifications affect genes that regulate responses to cocaine. We focused on Auts2, a gene linked to human evolution and cognitive disorders, which displays strong clustering of cocaine-induced chromatin modifications in this brain region. METHODS: We combined chromosome conformation capture, circularized chromosome conformation capture, and related approaches with behavioral paradigms relevant to cocaine phenotypes. Cell type-specific functions were assessed by fluorescence-activated cell sorting and viral-mediated overexpression in Cre-dependent mouse lines. RESULTS: We observed that Auts2 gene expression is increased by repeated cocaine administration specifically in D2-type medium spiny neurons in the nucleus accumbens, an effect seen in male but not female mice. Auts2 messenger RNA expression was also upregulated postmortem in the nucleus accumbens of male human cocaine addicts. We obtained evidence that chromosomal looping, bypassing 1524 kb of linear genome, connects Auts2 to the Caln1 gene locus under baseline conditions. This looping was disrupted after repeated cocaine exposure, resulting in increased expression of both genes in D2-type medium spiny neurons. Cocaine exposure reduces binding of CCCTC-binding factor, a chromosomal scaffolding protein, and increases histone and DNA methylation at the Auts-Caln1 loop base in the nucleus accumbens. Cell type-specific overexpression of Auts2 or Caln1 in D2-type medium spiny neurons demonstrated that both genes promote cocaine reward. CONCLUSIONS: These findings suggest that cocaine-induced alterations of neuronal three-dimensional genome organization destabilize higher order chromatin at specific loci that regulate responses to the drug.

A deletion at the mouse Xist gene exposes trans-effects that alter the heterochromatin of the inactive X chromosome and the replication time and DNA stability of both X chromosomes.

The inactive X chromosome of female mammals displays several properties of heterochromatin including late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, and methylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in female mouse fibroblasts led to the appearance of two histone modifications throughout the inactive X chromosome usually associated with euchromatin: histone H4 acetylation and histone H3 lysine-4 methylation. Despite these euchromatic properties, the inactive X chromosome was replicated even later in S phase than in wild-type female cells. Homozygosity for the deletion also caused regions of the active X chromosome that are associated with very high concentrations of LINE-1 elements to be replicated very late in S phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNA of both X chromosomes, leading to deletions and translocations. This was accompanied by the phosphorylation of p53 at serine-15, an event that occurs in response to DNA damage, and the accumulation of gamma-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains the DNA stability of both X chromosomes.

Monoallelic expression and asynchronous replication of p120 catenin in mouse and human cells.

The number of autosomal mammalian genes subject to random monoallelic expression has been limited to genes highly specific to the function of chemosensory neurons or lymphocytes, making this phenomenon difficult to address systematically. Here we demonstrate that asynchronous DNA replication can be used as a marker for the identification of novel genes with monoallelic expression and identify p120 catenin, a gene involved in cell adhesion, as belonging to this class. p120 is widely expressed; its presence in available cell lines allowed us to address quantitative aspects of monoallelic expression. We show that the epigenetic choice of active allele is clonally stable and that biallelic clones express p120 at twice the level of monoallelic clones. Unlike previous reports about genes of this type, we found that expression of p120 can be monoallelic in one cell type and strictly biallelic in another. We show that in human lymphoblasts, the silencing of one allele is incomplete. These unexpected properties are likely to be wide-spread, as we show that the Tlr4 gene shares them. Identification of monoallelic expression of a nearly ubiquitous gene indicates that this type of gene regulation is more common than previously thought. This has important implications for carcinogenesis and definition of cell identity.

Mice cloned from olfactory sensory neurons.

Cloning by nuclear transplantation has been successfully carried out in various mammals, including mice. Until now mice have not been cloned from post-mitotic cells such as neurons. Here, we have generated fertile mouse clones derived by transferring the nuclei of post-mitotic, olfactory sensory neurons into oocytes. These results indicate that the genome of a post-mitotic, terminally differentiated neuron can re-enter the cell cycle and be reprogrammed to a state of totipotency after nuclear transfer. Moreover, the pattern of odorant receptor gene expression and the organization of odorant receptor genes in cloned mice was indistinguishable from wild-type animals, indicating that irreversible changes to the DNA of olfactory neurons do not accompany receptor gene choice.

Biallelic germline transcription at the kappa immunoglobulin locus.

Rearrangement of antigen receptor genes generates a vast array of antigen receptors on lymphocytes. The establishment of allelic exclusion in immunoglobulin genes requires differential treatment of the two sequence identical alleles. In the case of the kappa immunoglobulin locus, changes in chromatin structure, methylation, and replication timing of the two alleles are all potentially involved in regulating rearrangement. Additionally, germline transcription of the kappa locus which precedes rearrangement has been proposed to reflect an opening of the chromatin structure rendering it available for rearrangement. As the initial restriction of rearrangement to one allele is critical to the establishment of allelic exclusion, a key question is whether or not germline transcription at the kappa locus is monoallelic or biallelic. We have used a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay and an RNA-fluorescence in situ hybridization (FISH) to show that germline transcription of the kappa locus is biallelic in wild-type immature B cells and in recombination activating gene (RAG)-/-, mu+ B cells. Therefore, germline transcription is unlikely to dictate which allele will be rearranged first and rather reflects a general opening on both alleles that must be accompanied by a mechanism allowing one of the two alleles to be rearranged first.