Genetic regulation of human brain proteome reveals proteins implicated in psychiatric disorders.

Psychiatric disorders are highly heritable yet polygenic, potentially involving hundreds of risk genes. Genome-wide association studies have identified hundreds of genomic susceptibility loci with susceptibility to psychiatric disorders; however, the contribution of these loci to the underlying psychopathology and etiology remains elusive. Here we generated deep human brain proteomics data by quantifying 11,608 proteins across 268 subjects using 11-plex tandem mass tag coupled with two-dimensional liquid chromatography-tandem mass spectrometry. Our analysis revealed 788 cis-acting protein quantitative trait loci associated with the expression of 883 proteins at a genome-wide false discovery rate <5%. In contrast to expression at the transcript level and complex diseases that are found to be mainly influenced by noncoding variants, we found protein expression level tends to be regulated by non-synonymous variants. We also provided evidence of 76 shared regulatory signals between gene expression and protein abundance. Mediation analysis revealed that for most (88%) of the colocalized genes, the expression levels of their corresponding proteins are regulated by cis-pQTLs via gene transcription. Using summary data-based Mendelian randomization analysis, we identified 4 proteins and 19 genes that are causally associated with schizophrenia. We further integrated multiple omics data with network analysis to prioritize candidate genes for schizophrenia risk loci. Collectively, our findings underscore the potential of proteome-wide linkage analysis in gaining mechanistic insights into the pathogenesis of psychiatric disorders.

Evaluating performance and applications of sample-wise cell deconvolution methods on human brain transcriptomic data.

Sample-wise deconvolution methods estimate cell-type proportions and gene expressions in bulk tissue samples, yet their performance and biological applications remain unexplored, particularly in human brain transcriptomic data. Here, nine deconvolution methods were evaluated with sample-matched data from bulk tissue RNA sequencing (RNA-seq), single-cell/nuclei (sc/sn) RNA-seq, and immunohistochemistry. A total of 1,130,767 nuclei per cells from 149 adult postmortem brains and 72 organoid samples were used. The results showed the best performance of dtangle for estimating cell proportions and bMIND for estimating sample-wise cell-type gene expressions. For eight brain cell types, 25,273 cell-type eQTLs were identified with deconvoluted expressions (decon-eQTLs). The results showed that decon-eQTLs explained more schizophrenia GWAS heritability than bulk tissue or single-cell eQTLs did alone. Differential gene expressions associated with Alzheimer's disease, schizophrenia, and brain development were also examined using the deconvoluted data. Our findings, which were replicated in bulk tissue and single-cell data, provided insights into the biological applications of deconvoluted data in multiple brain disorders.

Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform.

Genetic interactions mediate the emergence of phenotype from genotype, but technologies for combinatorial genetic perturbation in mammalian cells are challenging to scale. Here, we identify background-independent paralog synthetic lethals from previous CRISPR genetic interaction screens, and find that the Cas12a platform provides superior sensitivity and assay replicability. We develop the in4mer Cas12a platform that uses arrays of four independent guide RNAs targeting the same or different genes. We construct a genome-scale library, Inzolia, that is ~30% smaller than a typical CRISPR/Cas9 library while also targeting ~4000 paralog pairs. Screens in cancer cells demonstrate discrimination of core and context-dependent essential genes similar to that of CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes. Importantly, the in4mer platform offers a fivefold reduction in library size compared to other genetic interaction methods, substantially reducing the cost and effort required for these assays.

Transcriptome-wide profiling of acute stress induced changes in ribosome occupancy level using external standards.

Acute cellular stress is known to induce a global reduction in mRNA translation through suppression of cap dependent translation. Selective translation in response to acute stress has been shown to play important roles in regulating the stress response. However, accurately profiling translational changes transcriptome-wide in response to acute cellular stress has been challenging. Commonly used data normalization methods operate on the assumption that any systematic shifts are experimental artifacts. Consequently, if applied to profiling acute cellular stress-induced mRNA translation changes, these methods are expected to produce biased estimates. To address this issue, we designed, produced, and evaluated a panel of 16 oligomers to serve as external standards for ribosome profiling studies. Using Sodium Arsenite treatment-induced oxidative stress in lymphoblastoid cell lines as a model system, we applied spike-in oligomers as external standards. We found our spike-in oligomers to display a strong linear correlation between the observed and the expected quantification, with small ratio compression at the lower concentration range. Using the expected fold changes constructed from spike-in controls, we found in our dataset that TMM normalization, a popular global scaling normalization approach, produced 87.5% false positives at a significant cutoff that is expected to produce only 10% false positive discoveries. In addition, TMM normalization produced a systematic shift of fold change by 3.25 fold. These results highlight the consequences of applying global scaling approaches to conditions that clearly violate their key assumptions. In contrast, we found RUVg normalization using spike-in oligomers as control genes recapitulated the expected stress induced global reduction of translation and resulted in little, if any, systematic shifts in the expected fold change. Our results clearly demonstrated the utility of our spike-in oligomers, both for constructing expected results as controls and for data normalization.

The impact of common variants on gene expression in the human brain: from RNA to protein to schizophrenia risk.

Background: The impact of genetic variants on gene expression has been intensely studied at the transcription level, yielding in valuable insights into the association between genes and the risk of complex disorders, such as schizophrenia (SCZ). However, the downstream impact of these variants and the molecular mechanisms connecting transcription variation to disease risk are not well understood. Results: We quantitated ribosome occupancy in prefrontal cortex samples of the BrainGVEX cohort. Together with transcriptomics and proteomics data from the same cohort, we performed cis-Quantitative Trait Locus (QTL) mapping and identified 3,253 expression QTLs (eQTLs), 1,344 ribosome occupancy QTLs (rQTLs), and 657 protein QTLs (pQTLs) out of 7,458 genes quantitated in all three omics types from 185 samples. Of the eQTLs identified, only 34% have their effects propagated to the protein level. Further analysis on the effect size of prefrontal cortex eQTLs identified from an independent dataset showed clear post-transcriptional attenuation of eQTL effects. To investigate the biological relevance of the attenuated eQTLs, we identified 70 expression-specific QTLs (esQTLs), 51 ribosome-occupancy-specific QTLs (rsQTLs), and 107 protein-specific QTLs (psQTLs). Five of these omics-specific QTLs showed strong colocalization with SCZ GWAS signals, three of them are esQTLs. The limited number of GWAS colocalization discoveries from omics-specific QTLs and the apparent prevalence of eQTL attenuation prompted us to take a complementary approach to investigate the functional relevance of attenuated eQTLs. Using S-PrediXcan we identified 74 SCZ risk genes, 34% of which were novel, and 67% of these risk genes were replicated in a MR-Egger test. Notably, 52 out of 74 risk genes were identified using eQTL data and 70% of these SCZ-risk-gene-driving eQTLs show little to no evidence of driving corresponding variations at the protein level. Conclusion: The effect of eQTLs on gene expression in the prefrontal cortex is commonly attenuated post-transcriptionally. Many of the attenuated eQTLs still correlate with SCZ GWAS signal. Further investigation is needed to elucidate a mechanistic link between attenuated eQTLs and SCZ disease risk.

Disease modeling and gene correction of LGMDR21 iPSCs elucidates the role of POGLUT1 in skeletal muscle maintenance, regeneration, and the satellite cell niche.

Autosomal recessive limb-girdle muscular dystrophy 21 (LGMDR21) is caused by pathogenic variants in protein O-glucosyltransferase 1 (POGLUT1), which is responsible for O-glucosylation of specific epidermal growth factor (EGF) repeats found in ∼50 mammalian proteins, including Notch receptors. Previous data from patient biopsies indicated that impaired Notch signaling, reduction of muscle stem cells, and accelerated differentiation are probably involved in disease etiopathology. Using patient induced pluripotent stem cells (iPSCs), their corrected isotypes, and control iPSCs, gene expression profiling indicated dysregulation of POGLUT1, NOTCH, muscle development, extracellular matrix (ECM), cell adhesion, and migration as involved pathways. They also exhibited reduced in vitro POGLUT1 enzymatic activity and NOTCH signaling as well as defective myogenesis, proliferation, migration and differentiation. Furthermore, in vivo studies demonstrated significant reductions in engraftment, muscle stem cell formation, PAX7 expression, and maintenance, along with an increased percentage of mislocalized PAX7+ cells in the interstitial space. Gene correction in patient iPSCs using CRISPR-Cas9 nickase led to the rescue of the main in vitro and in vivo phenotypes. These results demonstrate the efficacy of iPSCs and gene correction in disease modeling and rescue of the phenotypes and provide evidence of the involvement of muscle stem cell niche localization, PAX7 expression, and cell migration as possible mechanisms in LGMDR21.

Evaluating performance and applications of sample-wise cell deconvolution methods on human brain transcriptomic data.

Sample-wise deconvolution methods have been developed to estimate cell-type proportions and gene expressions in bulk-tissue samples. However, the performance of these methods and their biological applications has not been evaluated, particularly on human brain transcriptomic data. Here, nine deconvolution methods were evaluated with sample-matched data from bulk-tissue RNAseq, single-cell/nuclei (sc/sn) RNAseq, and immunohistochemistry. A total of 1,130,767 nuclei/cells from 149 adult postmortem brains and 72 organoid samples were used. The results showed the best performance of dtangle for estimating cell proportions and bMIND for estimating sample-wise cell-type gene expression. For eight brain cell types, 25,273 cell-type eQTLs were identified with deconvoluted expressions (decon-eQTLs). The results showed that decon-eQTLs explained more schizophrenia GWAS heritability than bulk-tissue or single-cell eQTLs alone. Differential gene expression associated with multiple phenotypes were also examined using the deconvoluted data. Our findings, which were replicated in bulk-tissue RNAseq and sc/snRNAseq data, provided new insights into the biological applications of deconvoluted data.

Efficient gene knockout and genetic interactions: the IN4MER CRISPR/Cas12a multiplex knockout platform.

Genetic interactions mediate the emergence of phenotype from genotype, but initial technologies for combinatorial genetic perturbation in mammalian cells suffer from inefficiency and are challenging to scale. Recent focus on paralog synthetic lethality in cancer cells offers an opportunity to evaluate different approaches and improve on the state of the art. Here we report a meta-analysis of CRISPR genetic interactions screens, identifying a candidate set of background-independent paralog synthetic lethals, and find that the Cas12a platform provides superior sensitivity and assay replicability. We demonstrate that Cas12a can independently target up to four genes from a single guide array, and we build on this knowledge by constructing a genome-scale library that expresses arrays of four guides per clone, a platform we call 'in4mer'. Our genome-scale human library, with only 49k clones, is substantially smaller than a typical CRISPR/Cas9 monogenic library while also targeting more than four thousand paralog pairs, triples, and quads. Proof of concept screens in four cell lines demonstrate discrimination of core and context-dependent essential genes similar to that of state-of-the-art CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes, a capability not offered by any extant library. Importantly, the in4mer platform offers a fivefold reduction in the number of clones required to assay genetic interactions, dramatically improving the cost and effort required for these studies.

The impact of genetic background and cell lineage on the level and pattern of gene expression in position effect variegation.

BACKGROUND: Chromatin-based transcriptional silencing is often described as a stochastic process, largely because of the mosaic expression observed in position effect variegation (PEV), where a euchromatic reporter gene is silenced in some cells as a consequence of juxtaposition with heterochromatin. High levels of variation in PEV phenotypes are commonly observed in reporter stocks. To ascertain whether background mutations are the major contributors to this variation, we asked how much of the variation is determined by genetic variants segregating in the population, examining both the level and pattern of expression using the fruit fly, Drosophila melanogaster, as the model. RESULTS: Using selective breeding of a fourth chromosome PEV reporter line, 39C-12, we isolated two inbred lines exhibiting contrasting degrees of variegation (A1: low expression, D1: high expression). Within each inbred population, remarkable similarity is observed in the degree of variegation: 90% of the variation between the two inbred lines in the degree of silencing can be explained by genotype. Further analyses suggest that this result reflects the combined effect of multiple independent trans-acting loci. While the initial observations are based on a PEV phenotype scored in the fly eye (hsp70-white reporter), similar degrees of silencing were observed using a beta-gal reporter scored across the whole fly. Further, the pattern of variegation becomes almost identical within each inbred line; significant pigment enrichment in the same quadrant of the eye was found for both A1 and D1 lines despite different degrees of expression. CONCLUSIONS: The results indicate that background genetic variants play the major role in determining the variable degrees of PEV commonly observed in laboratory stocks. Interestingly, not only does the degree of variegation become consistent in inbred lines, the patterns of variegation also appear similar. Combining these observations with the spreading model for local heterochromatin formation, we propose an augmented stochastic model to describe PEV in which the genetic background drives the overall level of silencing, working with the cell lineage-specific regulatory environment to determine the on/off probability at the reporter locus in each cell. This model acknowledges cell type-specific events in the context of broader genetic impacts on heterochromatin formation.

Chromatin-mediated translational control is essential for neural cell fate specification.

Neural cell fate specification is a multistep process in which stem cells undergo sequential changes in states, giving rise to particular lineages such as neurons and astrocytes. This process is accompanied by dynamic changes of chromatin and in transcription, thereby orchestrating lineage-specific gene expression programs. A pressing question is how these events are interconnected to sculpt cell fate. We show that altered chromatin due to loss of the chromatin remodeler Chd5 causes neural stem cell activation to occur ahead of time. This premature activation is accompanied by transcriptional derepression of ribosomal subunits, enhanced ribosome biogenesis, and increased translation. These untimely events deregulate cell fate decisions, culminating in the generation of excessive numbers of astrocytes at the expense of neurons. By monitoring the proneural factor Mash1, we further show that translational control is crucial for appropriate execution of cell fate specification, thereby providing new insight into the interplay between transcription and translation at the initial stages of neurogenesis.

A Myogenic Double-Reporter Human Pluripotent Stem Cell Line Allows Prospective Isolation of Skeletal Muscle Progenitors.

Myogenic differentiation of human pluripotent stem cells (hPSCs) has been done by gene overexpression or directed differentiation. However, viral integration, long-term culture, and the presence of unwanted cells are the main obstacles. By using CRISPR/Cas9n, a double-reporter human embryonic stem cell (hESC) line was generated for PAX7/MYF5, allowing prospective readout. This strategy allowed pathway screen to define efficient myogenic induction in hPSCs. Next, surface marker screen allowed identification of CD10 and CD24 for purification of myogenic progenitors and exclusion of non-myogenic cells. CD10 expression was also identified on human satellite cells and skeletal muscle progenitors. In vitro and in vivo studies using transgene and/or reporter-free hPSCs further validated myogenic potential of the cells by formation of new fibers expressing human dystrophin as well as donor-derived satellite cells in NSG-mdx4Cv mice. This study provides biological insights for myogenic differentiation of hPSCs using a double-reporter cell resource and defines an improved myogenic differentiation and purification strategy.

Incompatibility of the circadian protein BMAL1 and HNF4α in hepatocellular carcinoma.

Hepatocyte nuclear factor 4 alpha (HNF4α) is a master regulator of liver-specific gene expression with potent tumor suppressor activity, yet many liver tumors express HNF4α. This study reveals that P1-HNF4α, the predominant isoform expressed in the adult liver, inhibits expression of tumor promoting genes in a circadian manner. In contrast, an additional isoform of HNF4α, driven by an alternative promoter (P2-HNF4α), is induced in HNF4α-positive human hepatocellular carcinoma (HCC). P2-HNF4α represses the circadian clock gene ARNTL (BMAL1), which is robustly expressed in healthy hepatocytes, and causes nuclear to cytoplasmic re-localization of P1-HNF4α. We reveal mechanisms underlying the incompatibility of BMAL1 and P2-HNF4α in HCC, and demonstrate that forced expression of BMAL1 in HNF4α-positive HCC prevents the growth of tumors in vivo. These data suggest that manipulation of the circadian clock in HNF4α-positive HCC could be a tractable strategy to inhibit tumor growth and progression in the liver.

Post-translational buffering leads to convergent protein expression levels between primates.

BACKGROUND: Differences in gene regulation between human and closely related species influence phenotypes that are distinctly human. While gene regulation is a multi-step process, the majority of research concerning divergence in gene regulation among primates has focused on transcription. RESULTS: To gain a comprehensive view of gene regulation, we surveyed genome-wide ribosome occupancy, which reflects levels of protein translation, in lymphoblastoid cell lines derived from human, chimpanzee, and rhesus macaque. We further integrated messenger RNA and protein level measurements collected from matching cell lines. We find that, in addition to transcriptional regulation, the major factor determining protein level divergence between human and closely related species is post-translational buffering. Inter-species divergence in transcription is generally propagated to the level of protein translation. In contrast, gene expression divergence is often attenuated post-translationally, potentially mediated through post-translational modifications. CONCLUSIONS: Results from our analysis indicate that post-translational buffering is a conserved mechanism that led to relaxation of selective constraint on transcript levels in humans.

Thousands of novel translated open reading frames in humans inferred by ribosome footprint profiling.

Accurate annotation of protein coding regions is essential for understanding how genetic information is translated into function. We describe riboHMM, a new method that uses ribosome footprint data to accurately infer translated sequences. Applying riboHMM to human lymphoblastoid cell lines, we identified 7273 novel coding sequences, including 2442 translated upstream open reading frames. We observed an enrichment of footprints at inferred initiation sites after drug-induced arrest of translation initiation, validating many of the novel coding sequences. The novel proteins exhibit significant selective constraint in the inferred reading frames, suggesting that many are functional. Moreover, ~40% of bicistronic transcripts showed negative correlation in the translation levels of their two coding sequences, suggesting a potential regulatory role for these novel regions. Despite known limitations of mass spectrometry to detect protein expressed at low level, we estimated a 14% validation rate. Our work significantly expands the set of known coding regions in humans.

Genomic variation. Impact of regulatory variation from RNA to protein.

The phenotypic consequences of expression quantitative trait loci (eQTLs) are presumably due to their effects on protein expression levels. Yet the impact of genetic variation, including eQTLs, on protein levels remains poorly understood. To address this, we mapped genetic variants that are associated with eQTLs, ribosome occupancy (rQTLs), or protein abundance (pQTLs). We found that most QTLs are associated with transcript expression levels, with consequent effects on ribosome and protein levels. However, eQTLs tend to have significantly reduced effect sizes on protein levels, which suggests that their potential impact on downstream phenotypes is often attenuated or buffered. Additionally, we identified a class of cis QTLs that affect protein abundance with little or no effect on messenger RNA or ribosome levels, which suggests that they may arise from differences in posttranslational regulation.

A distinct type of heterochromatin at the telomeric region of the Drosophila melanogaster Y chromosome.

Heterochromatin assembly and its associated phenotype, position effect variegation (PEV), provide an informative system to study chromatin structure and genome packaging. In the fruit fly Drosophila melanogaster, the Y chromosome is entirely heterochromatic in all cell types except the male germline; as such, Y chromosome dosage is a potent modifier of PEV. However, neither Y heterochromatin composition, nor its assembly, has been carefully studied. Here, we report the mapping and characterization of eight reporter lines that show male-specific PEV. In all eight cases, the reporter insertion sites lie in the telomeric transposon array (HeT-A and TART-B2 homologous repeats) of the Y chromosome short arm (Ys). Investigations of the impact on the PEV phenotype of mutations in known heterochromatin proteins (i.e., modifiers of PEV) show that this Ys telomeric region is a unique heterochromatin domain: it displays sensitivity to mutations in HP1a, EGG and SU(VAR)3-9, but no sensitivity to Su(z)2 mutations. It appears that the endo-siRNA pathway plays a major targeting role for this domain. Interestingly, an ectopic copy of 1360 is sufficient to induce a piRNA targeting mechanism to further enhance silencing of a reporter cytologically localized to the Ys telomere. These results demonstrate the diversity of heterochromatin domains, and the corresponding variation in potential targeting mechanisms.

Drosophila Piwi functions downstream of piRNA production mediating a chromatin-based transposon silencing mechanism in female germ line.

Transposon control is a critical process during reproduction. The PIWI family proteins can play a key role, using a piRNA-mediated slicing mechanism to suppress transposon activity posttranscriptionally. In Drosophila melanogaster, Piwi is predominantly localized in the nucleus and has been implicated in heterochromatin formation. Here, we use female germ-line-specific depletion to study Piwi function. This depletion of Piwi leads to infertility and to axis specification defects in the developing egg chambers; correspondingly, widespread loss of transposon silencing is observed. Germ-line Piwi does not appear to be required for piRNA production. Instead, Piwi requires Aubergine (and presumably secondary piRNA) for proper localization. A subset of transposons that show significant overexpression in germ-line Piwi-depleted ovaries exhibit a corresponding loss of HP1a and H3K9me2. Germ-line HP1a depletion also leads to a loss of transposon silencing, demonstrating the functional requirement for HP1a enrichment at these loci. Considering our results and those of others, we infer that germ-line Piwi functions downstream of piRNA production to promote silencing of some transposons via recruitment of HP1a. Thus, in addition to its better-known function in posttranscriptional silencing, piRNA also appears to function in a targeting mechanism for heterochromatin formation mediated by Piwi.

Identification of novel chromatin-associated proteins involved in programmed genome rearrangements in Tetrahymena.

Extensive DNA rearrangements occur during the differentiation of the developing somatic macronuclear genome from the germ line micronuclear genome of Tetrahymena thermophila. To identify genes encoding proteins likely to be involved in this process, we devised a cytological screen to find proteins that specifically localize in macronuclear anlagen (Lia proteins) at the stage when rearrangements occur. We compared the localization of these with that of the chromodomain protein, Pdd1p, which is the most abundant known participant in this genome reorganization. We show that in live cells, Pdd1p exhibits dynamic localization, apparently shuttling from the parental to the developing nuclei through cytoplasmic bodies called conjusomes. Visualization of GFP-tagged Pdd1p also highlights the substantial three-dimensional nuclear reorganization in the formation of nuclear foci that occur coincident with DNA rearrangements. We found that late in macronuclear differentiation, four of the newly identified proteins are organized into nuclear foci that also contain Pdd1p. These Lia proteins are encoded by primarily novel genes expressed at the beginning of macronuclear differentiation and have properties or recognizable domains that implicate them in chromatin or nucleic acid binding. Three of the Lia proteins also localize to conjusomes, a result that further implicates this structure in the regulation of DNA rearrangement.