Tissue of origin detection for cancer tumor using low-depth cfDNA samples through combination of tumor-specific methylation atlas and genome-wide methylation density in graph convolutional neural networks.

BACKGROUND: Cell free DNA (cfDNA)-based assays hold great potential in detecting early cancer signals yet determining the tissue-of-origin (TOO) for cancer signals remains a challenging task. Here, we investigated the contribution of a methylation atlas to TOO detection in low depth cfDNA samples. METHODS: We constructed a tumor-specific methylation atlas (TSMA) using whole-genome bisulfite sequencing (WGBS) data from five types of tumor tissues (breast, colorectal, gastric, liver and lung cancer) and paired white blood cells (WBC). TSMA was used with a non-negative least square matrix factorization (NNLS) deconvolution algorithm to identify the abundance of tumor tissue types in a WGBS sample. We showed that TSMA worked well with tumor tissue but struggled with cfDNA samples due to the overwhelming amount of WBC-derived DNA. To construct a model for TOO, we adopted the multi-modal strategy and used as inputs the combination of deconvolution scores from TSMA with other features of cfDNA. RESULTS: Our final model comprised of a graph convolutional neural network using deconvolution scores and genome-wide methylation density features, which achieved an accuracy of 69% in a held-out validation dataset of 239 low-depth cfDNA samples. CONCLUSIONS: In conclusion, we have demonstrated that our TSMA in combination with other cfDNA features can improve TOO detection in low-depth cfDNA samples.

Co-expression in tissue-specific gene networks links genes in cancer-susceptibility loci to known somatic driver genes.

BACKGROUND: The genetic background of cancer remains complex and challenging to integrate. Many somatic mutations within genes are known to cause and drive cancer, while genome-wide association studies (GWAS) of cancer have revealed many germline risk factors associated with cancer. However, the overlap between known somatic driver genes and positional candidate genes from GWAS loci is surprisingly small. We hypothesised that genes from multiple independent cancer GWAS loci should show tissue-specific co-regulation patterns that converge on cancer-specific driver genes. RESULTS: We studied recent well-powered GWAS of breast, prostate, colorectal and skin cancer by estimating co-expression between genes and subsequently prioritising genes that show significant co-expression with genes mapping within susceptibility loci from cancer GWAS. We observed that the prioritised genes were strongly enriched for cancer drivers defined by COSMIC, IntOGen and Dietlein et al. The enrichment of known cancer driver genes was most significant when using co-expression networks derived from non-cancer samples of the relevant tissue of origin. CONCLUSION: We show how genes within risk loci identified by cancer GWAS can be linked to known cancer driver genes through tissue-specific co-expression networks. This provides an important explanation for why seemingly unrelated sets of genes that harbour either germline risk factors or somatic mutations can eventually cause the same type of disease.

Partitioning and aggregating cross-tissue and tissue-specific genetic effects to identify gene-trait associations.

TWAS have shown great promise in extending GWAS loci to a functional understanding of disease mechanisms. In an effort to fully unleash the TWAS and GWAS information, we propose MTWAS, a statistical framework that partitions and aggregates cross-tissue and tissue-specific genetic effects in identifying gene-trait associations. We introduce a non-parametric imputation strategy to augment the inaccessible tissues, accommodating complex interactions and non-linear expression data structures across various tissues. We further classify eQTLs into cross-tissue eQTLs and tissue-specific eQTLs via a stepwise procedure based on the extended Bayesian information criterion, which is consistent under high-dimensional settings. We show that MTWAS significantly improves the prediction accuracy across all 47 tissues of the GTEx dataset, compared with other single-tissue and multi-tissue methods, such as PrediXcan, TIGAR, and UTMOST. Applying MTWAS to the DICE and OneK1K datasets with bulk and single-cell RNA sequencing data on immune cell types showcases consistent improvements in prediction accuracy. MTWAS also identifies more predictable genes, and the improvement can be replicated with independent studies. We apply MTWAS to 84 UK Biobank GWAS studies, which provides insights into disease etiology.

Expression genome-wide association study identifies key regulatory variants enriched with metabolic and immune functions in four porcine tissues.

BACKGROUND: Integration of high throughput DNA genotyping and RNA-sequencing data enables the discovery of genomic regions that regulate gene expression, known as expression quantitative trait loci (eQTL). In pigs, efforts to date have been mainly focused on purebred lines for traits with commercial relevance as such growth and meat quality. However, little is known on genetic variants and mechanisms associated with the robustness of an animal, thus its overall health status. Here, the liver, lung, spleen, and muscle transcriptomes of 100 three-way crossbred female finishers were studied, with the aim of identifying novel eQTL regulatory regions and transcription factors (TFs) associated with regulation of porcine metabolism and health-related traits. RESULTS: An expression genome-wide association study with 535,896 genotypes and the expression of 12,680 genes in liver, 13,310 genes in lung, 12,650 genes in spleen, and 12,595 genes in muscle resulted in 4,293, 10,630, 4,533, and 6,871 eQTL regions for each of these tissues, respectively. Although only a small fraction of the eQTLs were annotated as cis-eQTLs, these presented a higher number of polymorphisms per region and significantly stronger associations with their target gene compared to trans-eQTLs. Between 20 and 115 eQTL hotspots were identified across the four tissues. Interestingly, these were all enriched for immune-related biological processes. In spleen, two TFs were identified: ERF and ZNF45, with key roles in regulation of gene expression. CONCLUSIONS: This study provides a comprehensive analysis with more than 26,000 eQTL regions identified that are now publicly available. The genomic regions and their variants were mostly associated with tissue-specific regulatory roles. However, some shared regions provide new insights into the complex regulation of genes and their interactions that are involved with important traits related to metabolism and immunity.

Self-supervised learning for characterising histomorphological diversity and spatial RNA expression prediction across 23 human tissue types.

As vast histological archives are digitised, there is a pressing need to be able to associate specific tissue substructures and incident pathology to disease outcomes without arduous annotation. Here, we learn self-supervised representations using a Vision Transformer, trained on 1.7 M histology images across 23 healthy tissues in 838 donors from the Genotype Tissue Expression consortium (GTEx). Using these representations, we can automatically segment tissues into their constituent tissue substructures and pathology proportions across thousands of whole slide images, outperforming other self-supervised methods (43% increase in silhouette score). Additionally, we can detect and quantify histological pathologies present, such as arterial calcification (AUROC = 0.93) and identify missing calcification diagnoses. Finally, to link gene expression to tissue morphology, we introduce RNAPath, a set of models trained on 23 tissue types that can predict and spatially localise individual RNA expression levels directly from H&E histology (mean genes significantly regressed = 5156, FDR 1%). We validate RNAPath spatial predictions with matched ground truth immunohistochemistry for several well characterised control genes, recapitulating their known spatial specificity. Together, these results demonstrate how self-supervised machine learning when applied to vast histological archives allows researchers to answer questions about tissue pathology, its spatial organisation and the interplay between morphological tissue variability and gene expression.

Multi-omic characterization of allele-specific regulatory variation in hybrid pigs.

Hybrid mapping is a powerful approach to efficiently identify and characterize genes regulated through mechanisms in cis. In this study, using reciprocal crosses of the phenotypically divergent Duroc and Lulai pig breeds, we perform a comprehensive multi-omic characterization of regulatory variation across the brain, liver, muscle, and placenta through four developmental stages. We produce one of the largest multi-omic datasets in pigs to date, including 16 whole genome sequenced individuals, as well as 48 whole genome bisulfite sequencing, 168 ATAC-Seq and 168 RNA-Seq samples. We develop a read count-based method to reliably assess allele-specific methylation, chromatin accessibility, and RNA expression. We show that tissue specificity was much stronger than developmental stage specificity in all of DNA methylation, chromatin accessibility, and gene expression. We identify 573 genes showing allele specific expression, including those influenced by parent-of-origin as well as allele genotype effects. We integrate methylation, chromatin accessibility, and gene expression data to show that allele specific expression can be explained in great part by allele specific methylation and/or chromatin accessibility. This study provides a comprehensive characterization of regulatory variation across multiple tissues and developmental stages in pigs.

Integrating muti-omics data to identify tissue-specific DNA methylation biomarkers for cancer risk.

The relationship between tissue-specific DNA methylation and cancer risk remains inadequately elucidated. Leveraging resources from the Genotype-Tissue Expression consortium, here we develop genetic models to predict DNA methylation at CpG sites across the genome for seven tissues and apply these models to genome-wide association study data of corresponding cancers, namely breast, colorectal, renal cell, lung, ovarian, prostate, and testicular germ cell cancers. At Bonferroni-corrected P < 0.05, we identify 4248 CpGs that are significantly associated with cancer risk, of which 95.4% (4052) are specific to a particular cancer type. Notably, 92 CpGs within 55 putative novel loci retain significant associations with cancer risk after conditioning on proximal signals identified by genome-wide association studies. Integrative multi-omics analyses reveal 854 CpG-gene-cancer trios, suggesting that DNA methylation at 309 distinct CpGs might influence cancer risk through regulating the expression of 205 unique cis-genes. These findings substantially advance our understanding of the interplay between genetics, epigenetics, and gene expression in cancer etiology.

The cacao gene atlas: a transcriptome developmental atlas reveals highly tissue-specific and dynamically-regulated gene networks in Theobroma cacao L.

BACKGROUND: Theobroma cacao, the cocoa tree, is a tropical crop grown for its highly valuable cocoa solids and fat which are the basis of a 200-billion-dollar annual chocolate industry. However, the long generation time and difficulties associated with breeding a tropical tree crop have limited the progress of breeders to develop high-yielding disease-resistant varieties. Development of marker-assisted breeding methods for cacao requires discovery of genomic regions and specific alleles of genes encoding important traits of interest. To accelerate gene discovery, we developed a gene atlas composed of a large dataset of replicated transcriptomes with the long-term goal of progressing breeding towards developing high-yielding elite varieties of cacao. RESULTS: We describe the creation of the Cacao Transcriptome Atlas, its global characterization and define sets of genes co-regulated in highly organ- and temporally-specific manners. RNAs were extracted and transcriptomes sequenced from 123 different tissues and stages of development representing major organs and developmental stages of the cacao lifecycle. In addition, several experimental treatments and time courses were performed to measure gene expression in tissues responding to biotic and abiotic stressors. Samples were collected in replicates (3-5) to enable statistical analysis of gene expression levels for a total of 390 transcriptomes. To promote wide use of these data, all raw sequencing data, expression read mapping matrices, scripts, and other information used to create the resource are freely available online. We verified our atlas by analyzing the expression of genes with known functions and expression patterns in Arabidopsis (ACT7, LEA19, AGL16, TIP13, LHY, MYB2) and found their expression profiles to be generally similar between both species. We also successfully identified tissue-specific genes at two thresholds in many tissue types represented and a set of genes highly conserved across all tissues. CONCLUSION: The Cacao Gene Atlas consists of a gene expression browser with graphical user interface and open access to raw sequencing data files as well as the unnormalized and CPM normalized read count data mapped to several cacao genomes. The gene atlas is a publicly available resource to allow rapid mining of cacao gene expression profiles. We hope this resource will be used to help accelerate the discovery of important genes for key cacao traits such as disease resistance and contribute to the breeding of elite varieties to help farmers increase yields.

Long-read RNA sequencing identifies region- and sex-specific C57BL/6J mouse brain mRNA isoform expression and usage.

Alternative splicing (AS) contributes to the biological heterogeneity between species, sexes, tissues, and cell types. Many diseases are either caused by alterations in AS or by alterations to AS. Therefore, measuring AS accurately and efficiently is critical for assessing molecular phenotypes, including those associated with disease. Long-read sequencing enables more accurate quantification of differentially spliced isoform expression than short-read sequencing approaches, and third-generation platforms facilitate high-throughput experiments. To assess differences in AS across the cerebellum, cortex, hippocampus, and striatum by sex, we generated and analyzed Oxford Nanopore Technologies (ONT) long-read RNA sequencing (lrRNA-Seq) C57BL/6J mouse brain cDNA libraries. From > 85 million reads that passed quality control metrics, we calculated differential gene expression (DGE), differential transcript expression (DTE), and differential transcript usage (DTU) across brain regions and by sex. We found significant DGE, DTE, and DTU across brain regions and that the cerebellum had the most differences compared to the other three regions. Additionally, we found region-specific differential splicing between sexes, with the most sex differences in DTU in the cortex and no DTU in the hippocampus. We also report on two distinct patterns of sex DTU we observed, sex-divergent and sex-specific, that could potentially help explain sex differences in the prevalence and prognosis of various neurological and psychiatric disorders in future studies. Finally, we built a Shiny web application for researchers to explore the data further. Our study provides a resource for the community; it underscores the importance of AS in biological heterogeneity and the utility of long-read sequencing to better understand AS in the brain.

Tissue-specific and endogenous protein labeling with split fluorescent proteins.

The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG211 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG21-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications.

HNF4A and HNF1A exhibit tissue specific target gene regulation in pancreatic beta cells and hepatocytes.

HNF4A and HNF1A encode transcription factors that are important for the development and function of the pancreas and liver. Mutations in both genes have been directly linked to Maturity Onset Diabetes of the Young (MODY) and type 2 diabetes (T2D) risk. To better define the pleiotropic gene regulatory roles of HNF4A and HNF1A, we generated a comprehensive genome-wide map of their binding targets in pancreatic and hepatic cells using ChIP-Seq. HNF4A was found to bind and regulate known (ACY3, HAAO, HNF1A, MAP3K11) and previously unidentified (ABCD3, CDKN2AIP, USH1C, VIL1) loci in a tissue-dependent manner. Functional follow-up highlighted a potential role for HAAO and USH1C as regulators of beta cell function. Unlike the loss-of-function HNF4A/MODY1 variant I271fs, the T2D-associated HNF4A variant (rs1800961) was found to activate AKAP1, GAD2 and HOPX gene expression, potentially due to changes in DNA-binding affinity. We also found HNF1A to bind to and regulate GPR39 expression in beta cells. Overall, our studies provide a rich resource for uncovering downstream molecular targets of HNF4A and HNF1A that may contribute to beta cell or hepatic cell (dys)function, and set up a framework for gene discovery and functional validation.

Circadian-driven tissue specificity is constrained under caloric restricted feeding conditions.

Tissue specificity is a fundamental property of an organ that affects numerous biological processes, including aging and longevity, and is regulated by the circadian clock. However, the distinction between circadian-affected tissue specificity and other tissue specificities remains poorly understood. Here, using multi-omics data on circadian rhythms in mice, we discovered that approximately 35% of tissue-specific genes are directly affected by circadian regulation. These circadian-affected tissue-specific genes have higher expression levels and are associated with metabolism in hepatocytes. They also exhibit specific features in long-reads sequencing data. Notably, these genes are associated with aging and longevity at both the gene level and at the network module level. The expression of these genes oscillates in response to caloric restricted feeding regimens, which have been demonstrated to promote longevity. In addition, aging and longevity genes are disrupted in various circadian disorders. Our study indicates that the modulation of circadian-affected tissue specificity is essential for understanding the circadian mechanisms that regulate aging and longevity at the genomic level.

Temporal dynamics of the multi-omic response to endurance exercise training.

Regular exercise promotes whole-body health and prevents disease, but the underlying molecular mechanisms are incompletely understood1-3. Here, the Molecular Transducers of Physical Activity Consortium4 profiled the temporal transcriptome, proteome, metabolome, lipidome, phosphoproteome, acetylproteome, ubiquitylproteome, epigenome and immunome in whole blood, plasma and 18 solid tissues in male and female Rattus norvegicus over eight weeks of endurance exercise training. The resulting data compendium encompasses 9,466 assays across 19 tissues, 25 molecular platforms and 4 training time points. Thousands of shared and tissue-specific molecular alterations were identified, with sex differences found in multiple tissues. Temporal multi-omic and multi-tissue analyses revealed expansive biological insights into the adaptive responses to endurance training, including widespread regulation of immune, metabolic, stress response and mitochondrial pathways. Many changes were relevant to human health, including non-alcoholic fatty liver disease, inflammatory bowel disease, cardiovascular health and tissue injury and recovery. The data and analyses presented in this study will serve as valuable resources for understanding and exploring the multi-tissue molecular effects of endurance training and are provided in a public repository ( https://motrpac-data.org/ ).

Noninvasive assessment of organ-specific and shared pathways in multi-organ fibrosis using T1 mapping.

Fibrotic diseases affect multiple organs and are associated with morbidity and mortality. To examine organ-specific and shared biologic mechanisms that underlie fibrosis in different organs, we developed machine learning models to quantify T1 time, a marker of interstitial fibrosis, in the liver, pancreas, heart and kidney among 43,881 UK Biobank participants who underwent magnetic resonance imaging. In phenome-wide association analyses, we demonstrate the association of increased organ-specific T1 time, reflecting increased interstitial fibrosis, with prevalent diseases across multiple organ systems. In genome-wide association analyses, we identified 27, 18, 11 and 10 independent genetic loci associated with liver, pancreas, myocardial and renal cortex T1 time, respectively. There was a modest genetic correlation between the examined organs. Several loci overlapped across the examined organs implicating genes involved in a myriad of biologic pathways including metal ion transport (SLC39A8, HFE and TMPRSS6), glucose metabolism (PCK2), blood group antigens (ABO and FUT2), immune function (BANK1 and PPP3CA), inflammation (NFKB1) and mitosis (CENPE). Finally, we found that an increasing number of organs with T1 time falling in the top quintile was associated with increased mortality in the population. Individuals with a high burden of fibrosis in ≥3 organs had a 3-fold increase in mortality compared to those with a low burden of fibrosis across all examined organs in multivariable-adjusted analysis (hazard ratio = 3.31, 95% confidence interval 1.77-6.19; P = 1.78 × 10-4). By leveraging machine learning to quantify T1 time across multiple organs at scale, we uncovered new organ-specific and shared biologic pathways underlying fibrosis that may provide therapeutic targets.

TAD border deletion at the Kit locus causes tissue-specific ectopic activation of a neighboring gene.

Topologically associated domains (TADs) restrict promoter-enhancer interactions, thereby maintaining the spatiotemporal pattern of gene activity. However, rearrangements of the TADs boundaries do not always lead to significant changes in the activity pattern. Here, we investigated the consequences of the TAD boundaries deletion on the expression of developmentally important genes encoding tyrosine kinase receptors: Kit, Kdr, Pdgfra. We used genome editing in mice to delete the TADs boundaries at the Kit locus and characterized chromatin folding and gene expression in pure cultures of fibroblasts, mast cells, and melanocytes. We found that although Kit is highly active in both mast cells and melanocytes, deletion of the TAD boundary between the Kit and Kdr genes results in ectopic activation only in melanocytes. Thus, the epigenetic landscape, namely the mutual arrangement of enhancers and actively transcribing genes, is important for predicting the consequences of the TAD boundaries removal. We also found that mice without a TAD border between the Kit and Kdr genes have a phenotypic manifestation of the mutation - a lighter coloration. Thus, the data obtained shed light on the principles of interaction between the 3D chromatin organization and epigenetic marks in the regulation of gene activity.

Transposon DNA sequences facilitate the tissue-specific gene transfer of circulating tumor DNA between human cells.

The exchange of genes between cells is known to play an important physiological and pathological role in many organisms. We show that circulating tumor DNA (ctDNA) facilitates cell-specific gene transfer between human cancer cells and explain part of the mechanisms behind this phenomenon. As ctDNA migrates into the nucleus, genetic information is transferred. Cell targeting and ctDNA integration require ERVL, SINE or LINE DNA sequences. Chemically manufactured AluSp and MER11C sequences replicated multiple myeloma (MM) ctDNA cell targeting and integration. Additionally, we found that ctDNA may alter the treatment response of MM and pancreatic cancer models. This study shows that retrotransposon DNA sequences promote cancer gene transfer. However, because cell-free DNA has been detected in physiological and other pathological conditions, our findings have a broader impact than just cancer. Furthermore, the discovery that transposon DNA sequences mediate tissue-specific targeting will open up a new avenue for the delivery of genes and therapies.

Seasonal tissue-specific gene expression in wild crown-of-thorns starfish reveals reproductive and stress-related transcriptional systems.

Animals are influenced by the season, yet we know little about the changes that occur in most species throughout the year. This is particularly true in tropical marine animals that experience relatively small annual temperature and daylight changes. Like many coral reef inhabitants, the crown-of-thorns starfish (COTS), well known as a notorious consumer of corals and destroyer of coral reefs, reproduces exclusively in the summer. By comparing gene expression in 7 somatic tissues procured from wild COTS sampled on the Great Barrier Reef, we identified more than 2,000 protein-coding genes that change significantly between summer and winter. COTS genes that appear to mediate conspecific communication, including both signalling factors released into the surrounding sea water and cell surface receptors, are up-regulated in external secretory and sensory tissues in the summer, often in a sex-specific manner. Sexually dimorphic gene expression appears to be underpinned by sex- and season-specific transcription factors (TFs) and gene regulatory programs. There are over 100 TFs that are seasonally expressed, 87% of which are significantly up-regulated in the summer. Six nuclear receptors are up-regulated in all tissues in the summer, suggesting that systemic seasonal changes are hormonally controlled, as in vertebrates. Unexpectedly, there is a suite of stress-related chaperone proteins and TFs, including HIFa, ATF3, C/EBP, CREB, and NF-κB, that are uniquely and widely co-expressed in gravid females. The up-regulation of these stress proteins in the summer suggests the demands of oogenesis in this highly fecund starfish affects protein stability and turnover in somatic cells. Together, these circannual changes in gene expression provide novel insights into seasonal changes in this coral reef pest and have the potential to identify vulnerabilities for targeted biocontrol.

Origins of tissue and cell-type specificity in mitochondrial DNA (mtDNA) disease.

Mutations of mitochondrial (mt)DNA are a major cause of morbidity and mortality in humans, accounting for approximately two thirds of diagnosed mitochondrial disease. However, despite significant advances in technology since the discovery of the first disease-causing mtDNA mutations in 1988, the comprehensive diagnosis and treatment of mtDNA disease remains challenging. This is partly due to the highly variable clinical presentation linked to tissue-specific vulnerability that determines which organs are affected. Organ involvement can vary between different mtDNA mutations, and also between patients carrying the same disease-causing variant. The clinical features frequently overlap with other non-mitochondrial diseases, both rare and common, adding to the diagnostic challenge. Building on previous findings, recent technological advances have cast further light on the mechanisms which underpin the organ vulnerability in mtDNA diseases, but our understanding is far from complete. In this review we explore the origins, current knowledge, and future directions of research in this area.

Housekeeping protein-coding genes interrogated with tissue and individual variations.

Housekeeping protein-coding genes are stably expressed genes in cells and tissues that are thought to be engaged in fundamental cellular biological functions. They are often utilized as normalization references in molecular biology research and are especially important in integrated bioinformatic investigations. Prior studies have examined human housekeeping protein-coding genes by analyzing various gene expression datasets. The inclusion of different tissue types significantly impacted the discovery of housekeeping genes. In this report, we investigated particularly individual human subject expression differences in protein-coding genes across different tissue types. We used GTEx V8 gene expression datasets obtained from more than 16,000 human normal tissue samples. Furthermore, the Gini index is utilized to investigate the expression variations of protein-coding genes between tissue and individual donor subjects. Housekeeping protein-coding genes found using Gini index profiles may vary depending on the tissue subtypes investigated, particularly given the diverse sample size collections across the GTEx tissue subtypes. We subsequently selected major tissues and identified subsets of housekeeping genes with stable expression levels among human donors within those tissues. In this work, we provide alternative sets of housekeeping protein-coding genes that show more consistent expression patterns in human subjects across major solid organs. Weblink: https://hpsv.ibms.sinica.edu.tw .

Isoform-resolved genome annotation enables mapping of tissue-specific betalain regulation in amaranth.

Betalains are coloring pigments produced in some families of the order Caryophyllales, where they replace anthocyanins as coloring pigments. While the betalain pathway itself is well studied, the tissue-specific regulation of the pathway remains mostly unknown. We enhance the high-quality Amaranthus hypochondriacus reference genome and produce a substantially more complete genome annotation, incorporating isoform details. We annotate betalain and anthocyanin pathway genes along with their regulators in amaranth and map the genetic control and tissue-specific regulation of the betalain pathway. Our improved genome annotation allowed us to identify causal mutations that lead to a knock-out of red betacyanins in natural accessions of amaranth. We reveal the tissue-specific regulation of flower color via a previously uncharacterized MYB transcription factor, AhMYB2. Downregulation of AhMYB2 in the flower leads to reduced expression of key betalain enzyme genes and loss of red flower color. Our improved amaranth reference genome represents the most complete genome of amaranth to date and is a valuable resource for betalain and amaranth research. High similarity of the flower betalain regulator AhMYB2 to anthocyanin regulators and a partially conserved interaction motif support the co-option of anthocyanin regulators for the betalain pathway as a possible reason for the mutual exclusiveness of the two pigments.