The ENCODE Imputation Challenge: a critical assessment of methods for cross-cell type imputation of epigenomic profiles.

A promising alternative to comprehensively performing genomics experiments is to, instead, perform a subset of experiments and use computational methods to impute the remainder. However, identifying the best imputation methods and what measures meaningfully evaluate performance are open questions. We address these questions by comprehensively analyzing 23 methods from the ENCODE Imputation Challenge. We find that imputation evaluations are challenging and confounded by distributional shifts from differences in data collection and processing over time, the amount of available data, and redundancy among performance measures. Our analyses suggest simple steps for overcoming these issues and promising directions for more robust research.

The EN-TEx resource of multi-tissue personal epigenomes & variant-impact models.

Understanding how genetic variants impact molecular phenotypes is a key goal of functional genomics, currently hindered by reliance on a single haploid reference genome. Here, we present the EN-TEx resource of 1,635 open-access datasets from four donors (∼30 tissues × âˆ¼15 assays). The datasets are mapped to matched, diploid genomes with long-read phasing and structural variants, instantiating a catalog of >1 million allele-specific loci. These loci exhibit coordinated activity along haplotypes and are less conserved than corresponding, non-allele-specific ones. Surprisingly, a deep-learning transformer model can predict the allele-specific activity based only on local nucleotide-sequence context, highlighting the importance of transcription-factor-binding motifs particularly sensitive to variants. Furthermore, combining EN-TEx with existing genome annotations reveals strong associations between allele-specific and GWAS loci. It also enables models for transferring known eQTLs to difficult-to-profile tissues (e.g., from skin to heart). Overall, EN-TEx provides rich data and generalizable models for more accurate personal functional genomics.

Scalable detection of technically challenging variants through modified next-generation sequencing.

BACKGROUND: Some clinically important genetic variants are not easily evaluated with next-generation sequencing (NGS) methods due to technical challenges arising from high- similarity copies (e.g., PMS2, SMN1/SMN2, GBA1, HBA1/HBA2, CYP21A2), repetitive short sequences (e.g., ARX polyalanine repeats, FMR1 AGG interruptions in CGG repeats, CFTR poly-T/TG repeats), and other complexities (e.g., MSH2 Boland inversions). METHODS: We customized our NGS processes to detect the technically challenging variants mentioned above with adaptations including target enrichment and bioinformatic masking of similar sequences. Adaptations were validated with samples of known genotypes. RESULTS: Our adaptations provided high-sensitivity and high-specificity detection for most of the variants and provided a high-sensitivity primary assay to be followed with orthogonal disambiguation for the others. The sensitivity of the NGS adaptations was 100% for all of the technically challenging variants. Specificity was 100% for those in PMS2, GBA1, SMN1/SMN2, and HBA1/HBA2, and for the MSH2 Boland inversion; 97.8%-100% for CYP21A2 variants; and 85.7% for ARX polyalanine repeats. CONCLUSIONS: NGS assays can detect technically challenging variants when chemistries and bioinformatics are jointly refined. The adaptations described support a scalable, cost-effective path to identifying all clinically relevant variants within a single sample.

Expanded encyclopaedias of DNA elements in the human and mouse genomes.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

NF90/ILF3 is a transcription factor that promotes proliferation over differentiation by hierarchical regulation in K562 erythroleukemia cells.

NF90 and splice variant NF110 are DNA- and RNA-binding proteins encoded by the Interleukin enhancer-binding factor 3 (ILF3) gene that have been established to regulate RNA splicing, stabilization and export. The roles of NF90 and NF110 in regulating transcription as chromatin-interacting proteins have not been comprehensively characterized. Here, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) identified 9,081 genomic sites specifically occupied by NF90/NF110 in K562 cells. One third of NF90/NF110 peaks occurred at promoters of annotated genes. NF90/NF110 occupancy colocalized with chromatin marks associated with active promoters and strong enhancers. Comparison with 150 ENCODE ChIP-seq experiments revealed that NF90/NF110 clustered with transcription factors exhibiting preference for promoters over enhancers (POLR2A, MYC, YY1). Differential gene expression analysis following shRNA knockdown of NF90/NF110 in K562 cells revealed that NF90/NF110 activates transcription factors that drive growth and proliferation (EGR1, MYC), while attenuating differentiation along the erythroid lineage (KLF1). NF90/NF110 associates with chromatin to hierarchically regulate transcription factors that promote proliferation and suppress differentiation.

The Transcription Factor COL12 Is a Substrate of the COP1/SPA E3 Ligase and Regulates Flowering Time and Plant Architecture.

The ambient light environment controls many aspects of plant development throughout a plant's life cycle. Such complex control is achieved because a key repressor of light signaling, the Arabidopsis (Arabidopsis thaliana) COP1/SPA E3 ubiquitin ligase causes the degradation of multiple regulators of endogenous developmental pathways. This includes the CONSTANS (CO) transcription factor that is responsible for photoperiodic control of flowering time. There are 16 CO-like proteins whose functions are only partly understood. Here, we show that 14 CO-like (COL) proteins bind CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and SUPPRESSOR OF PHYTOCHROME A-105 (SPA)1 in vitro. We subsequently focused on COL12 and show that COL12 binds COP1 and SPA proteins in vivo. The COL12 protein is degraded in darkness in a COP1-dependent fashion, indicating that COL12 is a substrate of the COP1/SPA ubiquitin ligase. Overexpression of COL12 causes late flowering specifically in long day conditions by decreasing the expression of FLOWERING LOCUS T This phenotype is genetically dependent on CO. Consistent with this finding, COL12 physically interacts with CO in vivo, suggesting that COL12 represses flowering by inhibiting CO protein function. We show that COL12 overexpression did not alter CO protein stability. It is therefore likely that COL12 represses the activity of CO rather than CO levels. Overexpression of COL12 also affects plant architecture by increasing the number of rosette branches and reducing inflorescence height. These phenotypes are CO independent. Hence, we suggest that COL12 affects plant development through CO-dependent and CO-independent mechanisms.

Transcriptome dynamics of the stomatal lineage: birth, amplification, and termination of a self-renewing population.

Developmental transitions can be described in terms of morphology and the roles of individual genes, but also in terms of global transcriptional and epigenetic changes. Temporal dissections of transcriptome changes, however, are rare for intact, developing tissues. We used RNA sequencing and microarray platforms to quantify gene expression from labeled cells isolated by fluorescence-activated cell sorting to generate cell-type-specific transcriptomes during development of an adult stem-cell lineage in the Arabidopsis leaf. We show that regulatory modules in this early lineage link cell types that had previously been considered to be under separate control and provide evidence for recruitment of individual members of gene families for different developmental decisions. Because stomata are physiologically important and because stomatal lineage cells exhibit exemplary division, cell fate, and cell signaling behaviors, this dataset serves as a valuable resource for further investigations of fundamental developmental processes.

Direct roles of SPEECHLESS in the specification of stomatal self-renewing cells.

Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In Arabidopsis, the initiation and proliferation of stomatal lineage cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). SPCH-driven asymmetric and self-renewing divisions allow flexibility in stomatal production and overall organ growth. How SPCH directs stomatal lineage cell behaviors, however, is unclear. Here, we improved the chromatin immunoprecipitation (ChIP) assay and profiled the genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH controls key regulators of cell fate and asymmetric cell divisions and modulates responsiveness to peptide and phytohormone-mediated intercellular communication. Our results delineate the molecular pathways that regulate an essential adult stem cell lineage in plants.

Induced and natural variation of promoter length modulates the photoperiodic response of FLOWERING LOCUS T.

FLOWERING LOCUS T (FT) regulates the floral transition in many plant species by integrating environmental seasonal signals and internal cues. Here we show that two interdependent regulatory regions are necessary and sufficient to convey photoperiod responsiveness to FT. While a minimal distance between the regulatory regions is required to fully suppress FT expression under short days, increased distance reduces promoter response to long days. Natural variation at FT creating promoter length differences is widespread, correlates with longitudinal and latitudinal clines and affects a promoter region physically interacting with both photoperiod control regions. Three major FT promoter variants correlate with differences in FT allele usage in F1 hybrids. We propose that FT variation in cis could be adaptive by conferring differences in FT transcriptional control ultimately translating to increased fitness.

Elevated levels of MYB30 in the phloem accelerate flowering in Arabidopsis through the regulation of FLOWERING LOCUS T.

In Arabidopsis thaliana, the R2R3 MYB-like transcription factor MYB30 is a positive regulator of the pathogen-induced hypersensitive response and of brassinosteroid and abscisic acid signaling. Here, we show that MYB30 expressed under the control of the strong phloem-specific SUC2 promoter accelerates flowering both in long and short days. Early flowering is mediated by elevated expression of flowering locus T (FT), which can be observed in the absence and presence of CONSTANS (CO), the main activator of FT. CO-independent activation by high MYB30 expression results in FT levels that remain below those observed in the wild-type plants, which show an additive CO-dependent activation. In contrast, twin sister of FT (TSF) is repressed in plants expressing high levels of MYB30 in the phloem. In transient assays, MYB30 and CO additively increase the activity of a reporter construct driven by a 1 kb FT promoter. Acceleration of flowering by MYB30 does not require the presence of salicylic acid and is independent of FLC. Taken together, increased levels of MYB30, which was reported to be induced in response to the perception of pathogens, can accelerate flowering and MYB30 may thus be a candidate to mediate cross-talk between gene networks involved in biotic stress perception and flowering time.

Tissue-specific expression of FLOWERING LOCUS T in Arabidopsis is maintained independently of polycomb group protein repression.

The Polycomb Group (PcG) pathway represses transcription through a mechanism conserved among plants and animals. PcG-mediated repression can determine spatial territories of gene expression, but it remains unclear whether PcG-mediated repression is a regulatory requirement for all targets. Here, we show the role of PcG proteins in the spatial regulation of FLOWERING LOCUS T (FT), a main activator of flowering in Arabidopsis thaliana exclusively expressed in the vasculature. Strikingly, the loss of PcG repression causes down-regulation of FT. In addition, our results show how the effect of PcG-mediated regulation differs for target genes and that, for FT expression, it relies primarily on tissue differentiation.

cis-Regulatory elements and chromatin state coordinately control temporal and spatial expression of FLOWERING LOCUS T in Arabidopsis.

Flowering time of summer annual Arabidopsis thaliana accessions is largely determined by the timing of FLOWERING LOCUS T (FT) expression in the leaf vasculature. To understand the complex interplay between activating and repressive inputs controlling flowering through FT, cis-regulatory sequences of FT were identified in this study. A proximal and an approximately 5-kb upstream promoter region containing highly conserved sequence blocks were found to be essential for FT activation by CONSTANS (CO). Chromatin-associated protein complexes add another layer to FT regulation. In plants constitutively overexpressing CO, changes in chromatin status, such as a decrease in binding of LIKE HETEROCHROMATIN PROTEIN1 (LHP1) and increased acetylation of H3K9 and K14, were observed throughout the FT locus, although these changes appear to be a consequence of FT upregulation and not a prerequisite for activation. Binding of LHP1 was required to repress enhancer elements located between the CO-controlled regions. By contrast, the distal and proximal promoter sequences required for FT activation coincide with locally LHP1 and H3K27me3 depleted chromatin, indicating that chromatin status facilitates the accessibility of transcription factors to FT. Therefore, distant regulatory regions are required for FT transcription, reflecting the complexity of its control and differences in chromatin status delimit functionally important cis-regulatory regions.

From decision to commitment: the molecular memory of flowering.

During the floral transition the shoot apical meristem changes its identity from a vegetative to an inflorescence state. This change in identity can be promoted by external signals, such as inductive photoperiod conditions or vernalization, and is accompanied by changes in expression of key developmental genes. The change in meristem identity is usually not reversible, even if the inductive signal occurs only transiently. This implies that at least some of the key genes must possess an intrinsic memory of the newly acquired expression state that ensures irreversibility of the process. In this review, we discuss different molecular scenarios that may underlie a molecular memory of gene expression.

Arabidopsis SPA proteins regulate photoperiodic flowering and interact with the floral inducer CONSTANS to regulate its stability.

The four-member SPA protein family of Arabidopsis acts in concert with the E3 ubiquitin ligase COP1 to suppress photomorphogenesis in dark-grown seedlings. Here, we demonstrate that SPA proteins are, moreover, essential for photoperiodic flowering. Mutations in SPA1 cause phyA-independent early flowering under short day (SD) but not long day (LD) conditions, and this phenotype is enhanced by additional loss of SPA3 and SPA4 function. These spa1 spa3 spa4 triple mutants flower at the same time in LD and SD, indicating that the SPA gene family is essential for the inhibition of flowering under non-inductive SD. Among the four SPA genes, SPA1 is necessary and sufficient for normal photoperiodic flowering. Early flowering of SD-grown spa mutant correlates with strongly increased FT transcript levels, whereas CO transcript levels are not altered. Epistasis analysis demonstrates that both early flowering and FT induction in spa1 mutants is fully dependent on CO. Consistent with this finding, SPA proteins interact physically with CO in vitro and in vivo, suggesting that SPA proteins regulate CO protein function. Domain mapping shows that the SPA1-CO interaction requires the CCT-domain of CO, but is independent of the B-box type Zn fingers of CO. We further show that spa1 spa3 spa4 mutants exhibit strongly increased CO protein levels, which are not caused by a change in CO gene expression. Taken together, our results suggest, that SPA proteins regulate photoperiodic flowering by controlling the stability of the floral inducer CO.